Sustained Release Composition of Prostacyclin

ABSTRACT

The present invention relates to sustained release compositions of prostacyclin, as well as uses thereof, in particular for the prevention and/or treatment of pulmonary arterial hypertension.

The present application claims priority from PCT Patent Application No.PCT/EP2012/065742 filed on Aug. 10, 2012, which claims priority fromEuropean Patent Application No. EP 11177411.3 filed on Aug. 12, 2011,European Patent Application No. EP 11178075.5 filed on Aug. 19, 2011,and European Patent Application No. EP 12165512.0 filed on Apr. 25,2012, the disclosures of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

It is noted that citation or identification of any document in thisapplication is not an admission that such document is available as priorart to the present invention.

Pulmonary arterial hypertension (PAH) is an increase in blood pressurein the pulmonary artery, pulmonary vein, or pulmonary capillaries,leading to shortness of breath, dizziness, fainting, and other symptoms,all of which are exacerbated by exertion. PAH is a severe disease with amarkedly decreased exercise tolerance and heart failure. It is an orphandisease with an incidence of about 2-3 per million per year and aprevalence of about 15 per million. Median survival of patients withuntreated PAH is in the range of 2-3 years from time of diagnosis, withthe cause of death usually being right ventricular failure.

Pulmonary arterial hypertension involves the vasoconstriction ortightening of blood vessels connected to and within the lungs. Overtime, fibrosis causes the affected blood vessels to become both stifferand thicker which further increases the blood pressure within the lungsand impairs their blood flow. In addition, the increased workload of theheart causes hypertrophy of the right ventricle which ultimately causesright heart failure. As the blood flowing through the lungs decreases,the left side of the heart receives less blood and thus oxygen supply isbelow the required level, especially during physical activity.

A number of agents have been introduced for the treatment of PAH ofwhich prostacyclins are commonly considered to be the most effective.One prostacyclin is Epoprostenol which is a synthetic prostacyclin andmarketed as Flolan® (GlaxoSmithKline). It is given to patients viacontinuous infusion and requires a semi-permanent central venouscatheter which can cause sepsis and thrombosis. Flolan® is unstable, andtherefore has to be kept on ice during administration. Since it has ahalf-life of only 3 to 5 minutes, the infusion has to be continuousnight and day and any interruption can be fatal. Thus, treatment of PAHwith Flolan® is a huge burden for the patient.

Therefore, there was a need to develop other prostanoids, as has beendescribed for example in U.S. Pat. No. 4,306,075A and EP159784B1. Onesuch prostaglandin is treprostinil with the trade name Remodulin®(United Therapeutics). The half-life of treprostinil is 4 hours buttreprostinil is still required to be administered as a continuoussubcutaneous infusion or continuous intravenous infusion via an infusionpump that the patient must wear at all times.

As continuous infusion is also required for treprostinil, anyinterruption of drug delivery can be fatal, and there is therefore aneed to develop porstacyclin compounds with an even longer duration ofaction than that of treprostinil.

Subcutaneous infusion of treprostinil is frequently painful to theextent that the patient cannot tolerate the pain and consequently themode of administration is switched to intravenous infusion. However, anincreased risk of sepsis with intravenous Remodulin® has been reported.

As subcutaneous infusion is associated with pain, there is a need fordeveloping a prostacyclin that can be administered by subcutaneousadministration but with reduced rates of pain.

Another prostacyclin, Iloprost (Ilomedin) which is marketed as Ventavis®(Baier), was the only inhaled form of prostacyclin approved for use inthe US and Europe, until the inhaled form of treprostinil was approvedby the FDA in July 2009 which is marketed under the trade name TYVASO®(United Therapeutics).

Inhaled prostacyclin suffers from the drawback of not providing fullyefficacious plasma levels of drug throughout the dosing period, makinginhaled therapy less desired in severe patients.

Prostacyclins are the standard treatment of PAH, particularly in moresevere patients. Although inhaled treprostinil is more convenient andwithout the strong pain that is frequently associated withsubcutaneously infused treprostinil, inhalation is considered to be lesseffective and therefore less often prescribed.

Therefore, there exists a need to provide a more efficacious andcomfortable prostacyclin treatment for patients.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

It is further noted that the invention does not intend to encompasswithin the scope of the invention any previously disclosed product,process of making the product or method of using the product, whichmeets the written description and enablement requirements of the USPTO(35 U.S.C. 112, first paragraph) or the EPO (Article 83 of the EPC),such that applicant(s) reserve the right to disclaim, and herebydisclose a disclaimer of, any previously described product, method ofmaking the product, or process of using the product.

SUMMARY OF THE INVENTION

This object is achieved with a pharmaceutical composition comprising aprostacyclin compound and optionally one or more pharmaceuticallyacceptable excipients, which is characterized by having a concentrationof the prostacyclin compound that is sufficient to maintain atherapeutically effective level of prostacyclin in blood plasma for atleast 12 hours after a single subcutaneous or intramuscular injection.

DETAILED DESCRIPTION OF EMBODIMENTS

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for purposes of clarity, many other elements which are conventional inthis art. Those of ordinary skill in the art will recognize that otherelements are desirable for implementing the present invention. However,because such elements are well known in the art, and because they do notfacilitate a better understanding of the present invention, a discussionof such elements is not provided herein.

The present invention will now be described in detail on the basis ofexemplary embodiments.

In a further embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, for usein the treatment and/or prevention of diseases wherein the concentrationof the prostacyclin compound is sufficient to maintain a therapeuticallyeffective level of prostacyclin in blood plasma for at least 12 hoursafter a single subcutaneous or intramuscular injection.

This object is further achieved with a pharmaceutical compositioncomprising a prostacyclin compound and optionally one or morepharmaceutically acceptable excipients which is characterized by havinga pharmacokinetic profile in vivo in a mammal, in particular in a human,with substantially no burst of the prostacyclin compound.

In a further embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, for usein the treatment and/or prevention of diseases wherein a prostacyclincompound which is characterized by having a pharmacokinetic profile invivo in a mammal, in particular in a human, with substantially no burstof the prostacyclin compound.

This object is further achieved with a pharmaceutical compositioncomprising a prostacyclin compound and optionally one or morepharmaceutically acceptable excipients which is characterized byexhibiting a peak to trough ratio of less than 5, such as less than 3 orless than 2.

In a further embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, for usein the treatment and/or prevention of diseases wherein the peak totrough ratio of the prostacyclin compound less than 5, such as less than3 or less than 2.

This object is further achieved with a pharmaceutical compositioncomprising a prostacyclin compound and optionally one or morepharmaceutically acceptable excipients, wherein the prostacyclincompound has an activity of <20%, preferably <10%, more preferably <5%of the activity of free prostacyclin.

In a further embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, for usein the treatment and/or prevention of diseases, wherein the prostacyclincompound has an activity of <20%, preferably <10%, more preferably <5%of the activity of free prostacyclin.

This object is further achieved with a pharmaceutical compositioncomprising a prostacyclin prodrug and optionally one or morepharmaceutically acceptable excipients which is characterized in thatafter subcutaneous or intramuscular administration of said prostacyclinprodrug more than 50% of the administered prostacyclin dose isreleasable within the blood compartment.

In a preferred embodiment, the promoiety of the prodrug comprises alinear or branched PEG moiety.

In a further embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, for usein the treatment and/or prevention of diseases, wherein aftersubcutaneous or intramuscular administration of said prostacyclinprodrug more than 50% of the administered prostacyclin dose isreleasable within the blood compartment.

The prostacyclin compounds and pharmaceutical compositions andpharmaceutical compositions for use of the present invention reduce thepain associated with subcutaneous injections due to a protective carriermoiety which inhibits binding of the prostacyclin to its receptors inthe subcutaneous tissue, while at the same time providing relativelyconstant prostacyclin levels in the blood plasma of the patient.

The object is also achieved with a prostacyclin compound and apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, whichwhen administered to a patient in need thereof is characterized byhaving an absorption of the compound that is sufficiently fast and therelease of prostacyclin from the compound is sufficiently slow, so thatthe subcutaneous exposure to free prostacyclin is minimized.

In a further embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, for usein the treatment and/or prevention of diseases, wherein the absorptionof the compound in a patient, preferably a human patient, issufficiently fast and the release of prostacyclin from the compound issufficiently slow, so that the subcutaneous exposure to freeprostacyclin is minimized.

The prostacyclin compounds and pharmaceutical compositions andpharmaceutical compositions for use of the present invention are to beadministered to mammals, in particular humans. In particular, they areadministered to patients, in particular human patient, in need oftreatment with prostacyclin.

Such prostacyclin compounds elicit less of the known adverseprostacyclin effects on tissue after administration (such as erythema,edema, fibrosis and hemorrhage) upon subcutaneous or intramuscularadministration than compared to subcutaneous or intramuscular infusionof the same amount of the corresponding free prostacyclin.

Within the present invention the terms are used having the meaning asfollows.

The terms “drug”, “biologically active molecule”, “biologically activemoiety”, “biologically active agent”, “active agent”, “active substance”and the like mean any substance which can affect any physical orbiochemical properties of a biological organism, including but notlimited to viruses, bacteria, fungi, plants, animals, and humans. Inparticular, as used herein, the terms include any substance intended fordiagnosis, cure, mitigation, treatment, or prevention of disease inorganisms, in particular humans or other animals, or to otherwiseenhance physical or mental well-being of organisms, in particular humansor animals. According to the present invention, prostacyclins are drugs.

“Total polymer content of a pharmaceutical composition” means the totalpolymer concentration after complete removal of prostacyclin from saidpharmaceutical composition.

As used herein the term “prostacyclin” is intended to mean abiologically active moiety, including non-prostanoid biologically activemoieties, capable of eliciting a therapeutic effect similar toprostacyclin, by stimulating prostanoid receptors, such as, but notlimited to, DP₁, DP₂, EP₁, EP₂, EP₃, EP₄, FP, IP and TP, such asepoprostenol, iloprost, treprostinil, beraprost, ONO-1301 andNS-304/ACT-293987/selexipag, prostacyclin conjugated to low-molecularweight PEG, wherein low-molecular-weight PEG has a molecular weightsmaller than 10 kDa, beraprost sodium, epoprostenol sodium, iloprost incombination with bosentan, iloprost in combination with sildenafilcitrate, treprostinil, pegylated treprostinil, treprostinildiethanolamine and treprostinil sodium,2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide,{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid,8-[1,4,5-triphenyl-1H-imidazol-2-yl-oxy]octanoic acid, isocarbacyclin,cicaprost,[4-[2-(1,1-Diphenylethylsulfanyl)-ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxy]-aceticacid N-methyl-d-glucamine,7,8-dihydro-5-(2-(1-phenyl-1-pyrid-3-yl-methiminoxy)-ethyl)-a-naphthyloxyaceticacid, (5-(2-diphenylmethyl aminocarboxy)-ethyl)-a-naphthyloxyaceticacid, 2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid,[3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid,bosentan, 17[alpha], 20-dimethyl-[DELTA]6,6a6a-carba PGI1,15-deoxy-16[alpha]-hydroxy-16[beta],20-dimethyl-[DELTA]6,6a-6a-carbaPGI1 and pentoxifylline (1-{5-oxohexyl}-3,7-dimethylxanthine). Theprostacyclin compound may be in the form of a prodrug, in which case thecompound to be released in plasma is the active prostacyclin which formsafter administration of the prodrug.

The term “prostacyclin compound” refers to free prostacyclin,prostacyclin conjugates, prostacyclin depots with non-covalently orcovalently bound prostacyclin, and prostacyclin prodrugs, includingpolymer carrier-linked prostacyclin prodrug.

Targeting moieties are moieties that when present in a molecule, such asfor example a prodrug, allow preferential localization of such largermolecule in specific target areas of the organism to which it has beenadministered. Such specific target areas might be organs, certain celltypes or subcellular compartments. “Preferential localization” meansthat at least 10%, preferably at least 20% and more preferably at least30% of the biologically active moieties administered to a patient reachsaid specific target areas.

Targeting moieties may be divided into 3 classes according to size:

-   -   small molecular targeting moieties, for example C-glucuronide,        cobalamin, vitamins such as folic acid (folate) and analogs and        derivatives, carbohydrates, bisphosphonates,        N-acetylgalactosamine,    -   peptides, for example bombesin, somatostatin, LHRH, EGF, VEGF,        hCG, fragments of luteinizing hormone (LH), octreotide,        vapreotide, lanreotide, R^(C)-3940 series, decapeptyl, lupron,        zoladex, cetrorelix, peptides or peptidomimetics containing the        NGR or RGD motifs or derived from these motifs such as CNGRC        (linear), GNGRG (cyclic), ACDC RGD CFCG (cyclic), CDCRGDCFC,        CNGRC (cyclic), CRGDCGG, CNGRC, or other peptides such as        ATWLPPR, thrombospondin (TSP)-1 mimetics, (RGD peptidomimetic),        CTTHWGFTLC, CGNKRTRGC, neuropeptide substance P, SSP, the Sar9,        Met(O2)11 analog of substance P, cholecystokinin (CCK),        corticotropin-releasing hormone/factor (CRH/CRF), dermorphin,        FGF-2 or basic fibroblast growth factor, galanin, melanopsin,        neurotensin,    -   and protein or macro-molecular targeting moieties, for example        IL-2, GM-CSF, TNF-a, transferrin, immunoglobulins,        acetylated-LDL, lactoferrin (Lf) (also called lactotransferrin)        and lactoferricin (Lcin), gambogic acid (GA), antibody fragments        and affinity scaffold proteins.

In principle, any ligand of a cell surface receptor may beadvantageously used as a targeting moiety. For instance, ATWLPPR peptideis a potent antagonist of VEGF; thrombospondin-1 (TSP-1) inducesapoptosis in endothelial cells, RGD-motif mimics block integrinreceptors, NGR-containing peptides inhibit aminopeptidase N, and cyclicpeptides containing the sequence of HWGF selectively inhibit MMP-2 andMMP-9. LyP-1 peptide specifically binds to tumor lymphatic vessels.Illustrative other ligands include peptide ligands identified fromlibrary screens, tumor cell-specific peptides, tumor cell-specificaptamers, tumor cell-specific carbohydrates, tumor cell-specificmonoclonal or polyclonal antibodies, Fab or scFv (i.e., a single chainvariable region) fragments of antibodies such as, for example, a Fabfragment of an antibody directed to EphA2 or other proteins specificallyexpressed or uniquely accessible on metastatic cancer cells, smallorganic molecules derived from combinatorial libraries, growth factors,such as EGF, FGF, insulin, and insulin-like growth factors, andhomologous polypeptides, somatostatin and its analogs, transferrin,lipoprotein complexes, bile salts, selecting, steroid hormones,Arg-Gly-Asp containing peptides, retinoids, various Galectins, δ-opioidreceptor ligands, cholecystokinin A receptor ligands, ligands specificfor angiotensin AT1 or AT2 receptors, peroxisome proliferator-activatedreceptor λ ligands, β-lactam antibiotics such as penicillin, smallorganic molecules including antimicrobial drugs, and other moleculesthat bind specifically to a receptor preferentially expressed on thesurface of tumor cells or on an infectious organism, antimicrobial andother drugs designed to fit into the binding pocket of a particularreceptor based on the crystal structure of the receptor or other cellsurface protein, ligands of tumor antigens or other moleculespreferentially expressed on the surface of tumor cells, or fragments ofany of these molecules. Examples of tumor-specific antigens that canfunction as targeting moieties include extracellular epitopes of amember of the ephrin family of proteins, such as EphA2. EphA2 expressionis restricted to cell-cell junctions in normal cells, but EpbA2 isdistributed over the entire cell surface in metastatic tumor cells.Thus, EphA2 on metastatic cells would be accessible for binding to, forexample, a Fab fragment of an antibody conjugated to an immunogen,whereas the protein would not be accessible for binding to the Fabfragment on normal cells, resulting in a targeting moiety specific formetastatic cancer cells.

Further examples for such targeting moieties are: FSH-33, allatostatin1, hepatocarcinoma targeting peptide, peptide GFE, anti-EGFR antibodiesand/or antibody fragments, in particular cetuximab, CendR, iRGD peptide(RGD-CendR hybrid peptide), small molecules, antibodies and/or antibodyfragments binding to cancer-specific epitopes like e.g. CEA,gastrin-releasing peptide receptors, somatostatin receptors, galaninreceptors, follicle-stimulating hormone receptors, p32 protein,fibroblast growth factor receptors, HepG2, epidermal growth factorreceptors, integrin αvβ6, neuropilin-1 receptor and VEGF receptors.

“Free form” of a drug refers to the drug in its unmodified,pharmacologically active form, such as after being released from acarrier-linked prodrug. “Free form” of a prostacyclin refers to aprostacyclin in its unmodified, pharmacologically active form, such asafter being released from a conjugate, carrier-linked prodrug, or depot.

As used herein, the term “prodrug” is intended to mean a prostacyclincompound that undergoes biotransformation before exhibiting itspharmacological effects. Prodrugs can thus be viewed as biologicallyactive moieties containing specialized non-toxic protective groups usedin a transient manner to alter or to eliminate undesirable properties inthe parent molecule. For instance, the prodrug may be a biohydrolyzableamide and biohydrolyzable ester and also encompasses a) compounds inwhich the biohydrolyzable functionality in such a prodrug is encompassedin the compound, and b) compounds which may be oxidized or reducedbiologically at a given functional group. Typical prodrugs may be acarrier-linked prodrug that contains a temporary linkage of a givenactive substance with a transient carrier group that produces improvedphysicochemical or pharmacokinetic properties and that can be easilyremoved in vivo, usually by a hydrolytic cleavage; a cascade prodrug forwhich the cleavage of the carrier group becomes effective only afterunmasking an activating group.

To enhance physicochemical or pharmacokinetic properties of a drug, suchas prostacyclin, in vivo said drug can be conjugated with a carrier. Ifthe drug is reversibly bound to a carrier and/or a linker, such systemis commonly assigned as “carrier-linked prodrug”. According to thedefinitions provided by IUPAC (as given underhttp://www.chem.qmul.ac.uk/iupac/medchem/ah.html, accessed on Mar. 7,2011), a carrier-linked prodrug is a prodrug that contains a temporarylinkage of a given active substance with a reversible carrier group thatproduces improved physicochemical or pharmacokinetic properties and thatcan be easily removed in vivo, usually by a hydrolytic cleavage.

The term “promoiety” refers to the part of the prodrug which is not theprostacyclin drug, thus meaning for example the carrier as well as thereversible prodrug linker moiety and/or one or more spacermoiety/moieties, if present.

The term “reversible prodrug linker” or “transient prodrug linkers”refers to a moiety which on its one end is attached to a prostacyclinthrough a reversible linkage and at another end is attached through apermanent bond to either a spacer moiety permanently attached to acarrier moiety or is directly attached through a permanent bond to acarrier moiety. Such reversible prodrug linkers are non-enzymaticallyhydrolytically degradable, i.e. cleavable, under physiologicalconditions (aqueous buffer at pH 7.4, 37° C.) with half-lives rangingfrom, for example, one hour to three months. Reversible linkages are,for example, aconityls, acetals, amides, carboxylic anhydrides, esters,imines, hydrazones, maleamic acid amides, ortho esters, phosphamides,phosphoesters, phosphosilyl esters, silyl esters, sulfonic esters,aromatic carbamates, and combinations thereof.

Permanent linkages are non-enzymatically hydrolytically degradable underphysiological conditions (aqueous buffer at pH 7.4, 37° C.) withhalf-lives of six months or longer, such as, for example, amides.

If, for example, a functional group is coupled to another functionalgroup, the resulting chemical structure is referred to as “linkage”. Forexample, the reaction of an amine group with a carboxyl group results inan amide linkage.

As used herein the term “substantially no burst” or “substantiallyburstless” (both terms are used interchangeably in the presentdescription) is intended to mean that upon administration of aprostacyclin compound to a mammal, in particular a human, the ratio ofthe peak concentration of a detectable prostacyclin compound in bloodplasma during the first 48 hours after administration, such assubcutaneous or intramuscular, to the lowest concentration of adetectable prostacyclin compound in blood plasma after the peakconcentration during the first 48 hours after administration is lessthan 2 (substantially no burst detectable), preferred less than 1.5 (noburst detectable).

As used herein the term “burst” is intended to mean that uponadministration of a prostacyclin compound, which may be a prodrug or anactive prostacyclin compound, to a mammal, in particular a human, theratio of the peak concentration of a detectable prostacyclin compound inblood plasma during the first 48 hours after administration, such assubcutaneous or intramuscular, to the lowest concentration of adetectable prostacyclin compound in blood plasma after the peakconcentration during the first 48 hours after administration is 2 orhigher.

A “therapeutically effective amount” of a prostacyclin compound as usedherein means an amount sufficient to cure, alleviate or partially arrestthe clinical manifestations of a given disease and its complications ina mammal, in particular a human. An amount adequate to accomplish thisis defined as “therapeutically effective amount”. Effective amounts foreach purpose will depend on the severity of the disease or injury aswell as the weight and general state of the subject. It will beunderstood that determining an appropriate dosage may be achieved usingroutine experimentation, by constructing a matrix of values and testingdifferent points in the matrix, which is all within the ordinary skillsof a trained physician.

As used herein the term “a hydrogel” is intended to mean athree-dimensional, hydrophilic or amphiphilic polymeric network capableof taking up large quantities of water. The networks are composed ofhomopolymers or copolymers, are insoluble due to the presence ofcovalent chemical or physical (ionic, hydrophobic interactions,entanglements) crosslinks. The crosslinks provide the network structureand physical integrity. Hydrogels exhibit a thermodynamic compatibilitywith water which allows them to swell in aqueous media. The chains ofthe network are connected in such a fashion that pores exist and that asubstantial fraction of these pores are of dimensions between 1 nm and1000 nm.

The term “gel” refers to a non-crosslinked, jelly-like polymer solution.

As used herein the term “a depot” is intended to mean a drug deliverysystem, typically injected as a subcutaneous or intramuscular injection,of a prostacyclin compound, capable of consistently releasing the activecompound over an extended period of time.

As used herein the term “a peak concentration” is intended to mean thehighest concentration obtained after administration of a prostacyclincompound.

As used herein the term “peak to trough ratio” is intended to mean theratio between the highest steady state plasma concentration of acompound administered to a mammal, in particular a human, and the loweststeady state plasma concentration of a prostacyclin compound within agiven period between administrations.

“Alkyl” means a straight-chain (linear, unbranched) or branched carbonchain (unsubstituted alkyl). Optionally, one or more hydrogen atom(s) ofan alkyl carbon may be replaced by a substituent as indicated herein. Ingeneral, a preferred alkyl is C₁₋₆ alkyl.

“C₁₋₄ alkyl” means an alkyl chain having 1 to 4 carbon atoms(unsubstituted C₁₋₄ alkyl), e.g. if present at the end of a molecule:methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyltert-butyl, or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—,—CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of a molecule are linked by thealkyl group (also referred to as C₁₋₄ alkylene). Optionally, one or morehydrogen atom(s) of a C₁₋₄ alkyl carbon may be replaced by a substituentas indicated herein. Accordingly, “C₁₋₅₀ alkyl” means an alkyl chainhaving 1 to 50 carbon atoms.

“C₁₋₆ alkyl” means an alkyl chain having 1-6 carbon atoms, e.g. ifpresent at the end of a molecule: C₁₋₄ alkyl, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl,or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —C(CH₂)—, —CH₂—CH₂—CH₂—,—CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of a molecule are linked by thealkyl group (also referred to as C₁₋₆ alkylene). One or more hydrogenatom(s) of a C₁₋₆ alkyl carbon may be replaced by a substituent asindicated herein. The terms C₁₋₁₅ alkyl or C₁₋₁₅ alkylene are definedaccordingly.

“C₂₋₆ alkenyl” means an alkenyl chain having 2 to 6 carbon atoms, e.g.if present at the end of a molecule: —CH═CH₂, —CH═CH—CH₃, —CH₂—CH═CH₂,—CH═CH—CH₂—CH₃, —CH═CH—CH═CH₂, or e.g. —CH═CH—, when two moieties of amolecule are linked by the alkenyl group. One or more hydrogen atom(s)of a C₂₋₆ alkenyl carbon may be replaced by a substituent as indicatedherein.

The term C₂₋₄ alkenyl is defined accordingly.

“C₂₋₆ alkynyl” means an alkynyl chain having 2 to 6 carbon atoms, e.g.if present at the end of a molecule: —C≡CH, —CH₂—C≡CH, CH₂—CH₂—C≡CH,CH₂—C≡C—CH₃, or e.g. —C≡C— when two moieties of a molecule are linked bythe alkynyl group. One or more hydrogen atom(s) of a C₂₋₆ alkynyl carbonmay be replaced by a substituent as indicated herein. The term C₂₋₄alkynyl is defined accordingly.

“C₂₋₅₀ alkenyl” means a branched or unbranched alkenyl chain having 2 to50 carbon atoms (unsubstituted C₂₋₅₀ alkenyl), e.g. if present at theend of a molecule: —CH═CH₂, —CH═CH—CH₃, —CH₂—CH═CH₂, —CH═CH—CH₂—CH₃,—CH═CH—CH═CH₂, or e.g. —CH═CH—, when two moieties of a molecule arelinked by the alkenyl group. Optionally, one or more hydrogen atom(s) ofa C₂₋₅₀ alkenyl carbon may be replaced by a substituent as furtherspecified. Accordingly, the term “alkenyl” relates to a carbon chainwith at least one carbon carbon double bond. Optionally, one or moretriple bonds may occur. The term “C₂₋₁₅ alkenyl” is defined accordingly.

“C₂₋₅₀ alkynyl” means a branched or unbranched alkynyl chain having 2 to50 carbon atoms (unsubstituted C₂₋₅₀ alkynyl), e.g. if present at theend of a molecule: —C≡CH, —CH₂—C≡CH, CH₂—CH₂—C≡CH, CH₂—C≡C—CH₃, or e.g.—C≡C— when two moieties of a molecule are linked by the alkynyl group.Optionally, one or more hydrogen atom(s) of a C₂₋₅₀ alkynyl carbon maybe replaced by a substituent as further specified. Accordingly, the term“alkynyl” relates to a carbon chain with at least one carbon triplebond. Optionally, one or more double bonds may occur.

“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 7 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated (unsubstituted C₃₋₇ cycloalkyl), e.g.cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,cycloheptyl. Optionally, one or more hydrogen atom(s) of a cycloalkylcarbon may be replaced by a substituent as indicated herein. The term“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” also includes bridgedbicycles like norbonane (norbonanyl) or norbonene (norbonenyl).Accordingly, “C₃₋₅ cycloalkyl” means a cycloalkyl having 3 to 5 carbonatoms. Accordingly, “C₃₋₁₀ cycloalkyl” means a cycloalkyl having 3 to 10carbon atoms.

“Halogen” means fluoro, chloro, bromo or iodo. It is generally preferredthat halogen is fluoro or chloro.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom (unsubstituted 4 to 7 membered heterocyclyl).For the sake of completeness it is indicated that in some embodiments ofthe present invention, 4 to 7 membered heterocyclyl has to fulfilladditional requirements. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine. Optionally, one ormore hydrogen atom(s) of a 4 to 7 membered heterocyclyl may be replacedby a substituent.

“8 to 11 membered heterobicyclyl” or “8 to 11 membered heterobicycle”means a heterocyclic system of two rings with 8 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom (unsubstituted 8 to 11membered heterobicyclyl). Examples for a 8 to 11 membered heterobicycleare indole, indoline, benzofuran, benzothiophene, benzoxazole,benzisoxazole, benzothiazole, benzisothiazole, benzimidazole,benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline,dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline,decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline,benzazepine, purine or pteridine. The term 8 to 11 memberedheterobicycle also includes spiro structures of two rings like1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles like8-aza-bicyclo[3.2.1]octane. The term “9 to 11 membered heterobicyclyl”or “9 to 11 membered heterobicycle” is defined accordingly.

The term “aliphatic” means fully saturated.

The term “interrupted” means that between two carbon atoms of, forexample, a linker or a spacer or at the respective end of the carbonchain between the respective carbon atom and the hydrogen atom a group(such a —O— or —NH—) is inserted.

In general the term “substituted” preferably refers to substituents,which are the same or different and which are independently selectedfrom the group consisting of halogen, CN, COOR^(b9), OR^(b9),C(O)R^(b9), C(O)N(R^(b9)R^(b9a)), S(O)₂N(R^(b9)R^(b9a)),S(O)N(R^(b9)R^(b9a)), S(O)₂R^(b9), S(O)R^(b9),N(R^(b9))S(O)₂N(R^(b9a)R^(b9b)), SR^(b9), N(R^(b9)R^(b9a)), NO₂,OC(O)R^(b9), N(R^(b9))C(O)R^(b9a), N(R^(b9))S(O)₂R^(b9a),N(R^(b9))S(O)R^(b9a), N(R^(b9))C(O)OR^(b9a),N(R^(b9))C(O)N(R^(b9a)R^(b9b)), OC(O)N(R^(b9)R^(b9a)), T^(b), C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl,

-   -   wherein T^(b), C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are        optionally substituted with one or more R^(b10), which are the        same or different, and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and        C₂₋₅₀ alkynyl are optionally interrupted by one or more groups        selected from the group consisting of T^(b), —C(O)O—; —O—;        —C(O)—; —C(O)N(R^(b11))—; —S(O)₂N(R^(b11))—; —S(O)N(R^(b11))—;        —S(O)₂—; —S(O)—; —N(R^(b11))S(O)₂N(R^(b11a))—; —S—;        —N(R^(b11))—; —OC(O)R^(b11); —N(R^(b11))C(O)—;        —N(R^(b11))S(O)₂—; —N(R^(b11))S(O)—; —N(R^(b11))C(O)O—;        —N(R^(b11))C(O)N(R^(b11a))—; and —OC(O)N(R^(b11)R^(b11a));    -   R^(b9), R^(b9a), R^(b9b) independently selected from the group        consisting of H; T^(b); and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and        C₂₋₅₀ alkynyl,        -   wherein T^(b), C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl            are optionally substituted with one or more R^(b10), which            are the same or different, and wherein C₁₋₅₀ alkyl; C₂₋₅₀            alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted by one            or more groups selected from the group consisting of T^(b),            —C(O)O—, —O—, —C(O)—, —C(O)N(R^(b11))—, —S(O)₂N(R^(b11))—,            —S(O)N(R^(b11))—, —S(O)₂—, —S(O)—,            —N(R^(b11))S(O)₂N(R^(b11a))—, —S—, —N(R^(b11))—,            —OC(O)R^(b11), —N(R^(b11))C(O)—, —N(R^(b11))S(O)₂—,            —N(R^(b11))S(O)—, —N(R^(b11))C(O)O—,            —N(R^(b11))C(O)N(R^(b11a))—, and —OC(O)N(R^(b11)R^(b11a)),        -   T^(b) is selected from the group consisting of phenyl,            naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4-            to 7-membered heterocyclyl, and 9- to 11-membered            heterobicyclyl, wherein T^(b) is optionally substituted with            one or more R^(b10), which are the same or different,        -   R^(b10) is halogen, CN, oxo (═O), COOR^(b12), OR^(b12),            C(O)R^(b12), C(O)N(R^(b12)R^(b12a)),            S(O)₂N(R^(b12)R^(b12a)), S(O)N(R^(b12)R^(b12a)),            S(O)₂R^(b12), S(O)R^(b12),            N(R^(b12))S(O)₂N(R^(b12a)R^(b12b)), SR^(b12),            N(R^(b12)R^(b12a)), NO₂, OC(O)R^(b12),            N(R^(b12))C(O)R^(b12a), N(R^(b12))S(O)₂R^(b12a),            N(R^(b12))S(O)R^(b12a), N(R^(b12))C(O)OR^(b12a),            N(R^(b12))C(O)N(R^(b12a)R^(b12b)), OC(O)N(R^(b12)R^(b12a)),            or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted            with one or more halogen, which are the same or different,        -   R^(b11), R^(b11a), R^(b12), R^(b12a), R^(b12b) are            independently selected from the group consisting of H; or            C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted            with one or more halogen, which are the same or different.

The term “interrupted” means that between two carbons a group isinserted or that at the end of the carbon chain between the carbon andhydrogen.

In general the term “comprise” or “comprising” also encompasses “consistof” or “consisting of”.

The term “pharmaceutically acceptable” means approved by a regulatoryagency such as the EMA (Europe) and/or the FDA (US) and/or any othernational regulatory agency for use in animals, preferably in humans.

“Pharmaceutical composition” or “composition” means a compositioncontaining one or more drugs or prodrugs, in particular a prostacyclincompound, and one or more inert ingredients, as well as any productwhich results, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition obtainable by admixing a water-soluble carrier-linkedprodrug of the present invention and a pharmaceutically acceptableexcipient.

“Dry composition” means that a pharmaceutical composition comprising aprostacyclin compound is provided in a dry form in a container. Suitablemethods for drying are spray-drying and lyophilization (freeze-drying).Such dry composition comprising a prostacyclin compound has a residualwater content of a maximum of 10%, preferably less than 5% and morepreferably less than 2% (determined according to Karl Fischer). Thepreferred method of drying is lyophilization. “Lyophilized composition”means that the pharmaceutical composition comprising a prostacyclincompound was first frozen and subsequently subjected to water reductionby means of reduced pressure. This terminology does not excludeadditional drying steps which occur in the manufacturing process priorto filling the composition into the final container.

“Lyophilization” (freeze-drying) is a dehydration process, characterizedby freezing a composition and then reducing the surrounding pressureand, optionally, adding heat to allow the frozen water in thecomposition to sublime directly from the solid phase to gas. Typically,the sublimed water is collected by desublimation.

“Reconstitution” means the addition of a liquid to bring back theoriginal form of a composition.

“Reconstitution solution” refers to the liquid used to reconstitute thedry composition of a pharmaceutical composition comprising aprostacyclin compound prior to administration to a patient in needthereof.

“Container” means any container in which the pharmaceutical compositioncomprising a prostacyclin compound is comprised and can be stored untilreconstitution.

A “therapeutically effective amount” of a prostacyclin compound as usedherein means an amount sufficient to cure, alleviate or partially arrestthe clinical manifestations of a given disease and its complications. Anamount adequate to accomplish this is defined as “therapeuticallyeffective amount”. Effective amounts for each purpose will depend on theseverity of the disease or injury as well as the weight and generalstate of the subject. It will be understood that determining anappropriate dosage may be achieved using routine experimentation, byconstructing a matrix of values and testing different points in thematrix, which is all within the ordinary skills of a trained physicianor veterinary.

“Buffer” or “buffering agent” refers to chemical compounds that maintainthe pH in a desired range. Physiologically tolerated buffers are, forexample, sodium phosphate, succinate, histidine, bicarbonate, citrateand acetate, sulphate, nitrate, chloride, pyruvate. Antacids such asMg(OH)₂ or ZnCO₃ may be also used. Buffering capacity may be adjusted tomatch the conditions most sensitive to pH stability.

“Excipients” refers to compounds administered together with thetherapeutic agent, for example, buffering agents, isotonicity modifiers,preservatives, stabilizers, anti-adsorption agents, oxidation protectionagents, or other auxiliary agents. However, in some cases, one excipientmay have dual or triple functions.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, significantly prevents or reduces chemical and/or physicalinstability of the protein upon drying in general and especially duringlyophilization and subsequent storage. Exemplary lyoprotectants includesugars, such as sucrose or trehalose; amino acids such as monosodiumglutamate or histidine; methylamines such as betaine; lyotropic saltssuch as magnesium sulfate; polyols such as trihydric or higher sugaralcohols, e.g. glycerin, erythritol, glycerol, arabitol, xylitol,sorbitol, and mannitol; ethylene glycol; propylene glycol; polyethyleneglycol; pluronics; hydroxyalkyl starches, e.g. hydroxyethyl starch(HES), and combinations thereof.

“Surfactant” refers to wetting agents that lower the surface tension ofa liquid.

“Isotonicity modifiers” refer to compounds which minimize pain that canresult from cell damage due to osmotic pressure differences at theinjection depot.

The term “stabilizers” refers to compounds used to stabilize thepolymeric prodrug. Stabilisation is achieved by strengthening of theprotein-stabilising forces, by destabilisation of the denatured state,or by direct binding of excipients to the protein.

“Anti-adsorption agents” refers to mainly ionic or non-ionic surfactantsor other proteins or soluble polymers used to coat or adsorbcompetitively to the inner surface of the composition's container.Chosen concentration and type of excipient depends on the effect to beavoided but typically a monolayer of surfactant is formed at theinterface just above the CMC value.

“Oxidation protection agents” refers to antioxidants such as ascorbicacid, ectoine, methionine, glutathione, monothioglycerol, morin,polyethylenimine (PEI), propyl gallate, vitamin E, chelating agents suchas citric acid, EDTA, hexaphosphate, thioglycolic acid.

“Antimicrobial” refers to a chemical substance that kills or inhibitsthe growth of microorganisms, such as bacteria, fungi, yeasts,protozoans and/or destroys viruses.

“Sealing a container” means that the container is closed in such waythat it is airtight, allowing no gas exchange between the outside andthe inside and keeping the content sterile.

“Pharmaceutically acceptable” is meant to encompass any excipient and/oradditive, which does not interfere with the effectiveness of thebiological activity of the active ingredient and that, is not toxic tothe host to which it is administered.

The term “reagent” refers to an intermediate or starting material usedin the assembly process of a molecule.

The term “derivatives” refers to chemical functional groups suitablysubstituted with protecting and/or activation groups or to activatedforms of a corresponding chemical functional group which are known tothe person skilled in the art. For example, activated forms of carboxylgroups include but are not limited to active esters, such assuccinimidyl ester, benzotriazyl ester, nitrophenyl ester,pentafluorophenyl ester, azabenzotriazyl ester, acyl halogenides, mixedor symmetrical anhydrides, acyl imidazole.

The term “non-enzymatically cleavable linker” refers to linkers that arehydrolytically degradable under physiological conditions withoutenzymatic activity.

“Non-biologically active linker” means a linker which does not show thepharmacological effects of the drug (D-H) derived from the biologicallyactive moiety.

The terms “spacer”, “spacer group”, “spacer molecule”, and “spacermoiety” are used interchangeably and refers to any moiety suitable forconnecting two moieties, such as C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl or C₂₋₅₀alkinyl, which fragment is optionally interrupted by one or more groupsselected from —NH—, —N(C₁₋₄ alkyl)-, —O—, —S—, —C(O)—, —C(O)NH—,—C(O)N(C₁₋₄ alkyl)-, —O—C(O)—, —S(O)—, —S(O)₂—, 4 to 7 memberedheterocyclyl, phenyl and naphthyl.

The phrases “in bound form” or “moiety” refer to sub-structures whichare part of a larger molecule. The phrase “in bound form” is used tosimplify reference to moieties by naming or listing reagents, startingmaterials or hypothetical starting materials well known in the art, andwhereby “in bound form” means that for example one or more hydrogenradicals (—H), or one or more activating or protecting groups present inthe reagents or starting materials are not present in the moiety.

“Continuous release” refers to an uninterrupted release of prostacyclincompound.

As used herein a single prostacyclin compound dose is given in mg andconcentration of a prostacyclin compound in a pharmaceutical compositionis given in mg/mL. If the prostacyclin compound is a prodrug, theconcentration is based on quantitative release of free prostacyclin fromsaid prodrug. By methods well-known in the art, aliquots of acomposition are subjected to prostacyclin-releasing conditions (aqueousbuffer pH 7.4, 37° C., or accelerated conditions at elevated pH), untilno significant increase in prostacyclin concentration is observed andthe total amount of released prostacyclin is determined. It isunderstood that in the case of soluble carriers, quantitative release issynonymous to quantitative hydrolysis.

“Activity” of a prostacyclin compound is measured by ADP andcollagen-Induced Aggregation of human and/or rat platelets. The effectsof prostacyclin compounds on human and rat platelet aggregation can beevaluated by methods known in the art. Generally, platelets are obtainedfrom healthy human volunteers or male Wistar rats. Prior to induction ofaggregation with either ADP or collagen, platelets are pre-incubatedwith the appropriate test article for 1 minute. Platelets are incubatedwith prodrugs of prostacyclin compounds and the free form of theprostacyclin in parallel at concentrations sufficient to determine IC₅₀.Prodrugs of prostacyclin compounds are considered inactive when the IC₅₀value is at least 20 fold higher compared to the IC₅₀ value of thecorresponding free form of prostacyclin.

The present invention relates to a pharmaceutical composition comprisinga prostacyclin compound, and optionally one or more pharmaceuticallyacceptable excipients, which is characterized by having a concentrationof the prostacyclin compound that is sufficient to maintain atherapeutically effective level of prostacyclin in blood plasma for atleast 12 hours after a single subcutaneous or intramuscular injection.

In a further embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound andoptionally one or more pharmaceutically acceptable excipients, for usein the treatment and/or prevention of diseases wherein the concentrationof the prostacyclin compound is sufficient to maintain a therapeuticallyeffective level of prostacyclin in blood plasma for at least 12 hoursafter a single subcutaneous or intramuscular injection.

Such a concentration will vary from subject to subject and depends onthe therapeutic window in an individual subject, but needs to besufficient in order for the therapeutic effect to be present during atleast 12 hours.

Preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized in that it comprises a prostacyclin compound in aconcentration of at least 0.05 mg/ml.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is characterizedin that it further comprises a prostacyclin compound in a concentrationof at least 0.05 mg/ml, and wherein a single dose of said pharmaceuticalcomposition comprises at least 0.05 mg of the prostacyclin compound.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound further exhibitsa time period between administration of a pharmaceutical composition ofat least about 12 hours, such as at least 16 hours, typically at leastone day.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound further exhibitsa pharmacokinetic profile in vivo in a mammal, in particular a humanwith substantially no burst of the prostacyclin compound.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized by exhibiting a peak to trough ratio in a mammal, inparticular a human of less than 5, such as less than 3, or less than 2.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized in that the prostacyclin compound has an activity of <20%,preferably <10%, more preferably <5% of the activity of freeprostacyclin.

It is understood that the activity of free prostacyclin is understood asthe activity of the corresponding free prostacyclin which freeprostacyclin is obtained after release from e.g. the prodrug or depot.

In addition, another aspect of the present invention relates to apharmaceutical composition or pharmaceutical composition for usecomprising a prostacyclin compound which is characterized by having apharmacokinetic profile in vivo in a mammal, in particular a human withsubstantially no burst of the prostacyclin compound.

Preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound exhibits a peakto trough ratio in a mammal, in particular a human of less than 5, suchas less than 3, or less than 2

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized in that the prostacyclin compound has an activity of <20%,preferably <10%, more preferably <5% of the activity of freeprostacyclin.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized by being administered to a mammal, preferably a human,more preferably a human patient by injection, such as subcutaneous orintramuscular injection.

The volume to be administered in order to administer to a mammal,preferably a human, more preferably a human patient an effective dose,for example by a syringe, to a subject, such as a human, is preferablyless than 3 ml, typically 2 ml or less.

In addition, another aspect of the present invention further relates toa pharmaceutical composition or pharmaceutical composition for usecomprising a prostacyclin compound in which the prostacyclin compoundexhibits a peak to trough ratio in a mammal, in particular a human ofless than 5, such as less than 3, or less than 2.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized in that the prostacyclin compound has an activity of <20%,preferably <10%, more preferably <5% of the activity of freeprostacyclin.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized by being administered to a mammal, preferably a human,more preferably a human patient by injection, such as subcutaneous orintramuscular.

In addition, another aspect of the present invention further relates toa pharmaceutical composition or pharmaceutical composition for usecomprising a prostacyclin compound wherein the prostacyclin compound hasan activity of <20%, preferably <10%, more preferably <5% of theactivity of free prostacyclin.

Even more preferably, such pharmaceutical composition or pharmaceuticalcomposition for use comprising a prostacyclin compound is furthercharacterized by being administered by injection, such as subcutaneousor intramuscular injection.

In another preferred embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound for use insubcutaneous or intramuscular injection.

In another preferred embodiment, the present invention relates to apharmaceutical composition comprising a prostacyclin compound, whereinsaid pharmaceutical composition is characterized in that theprostacyclin compound releases prostacyclin in a plasma-independentmanner. Preferably, the prostacyclin compound releases prostacyclin inan enzyme-independent manner.

The term “plasma-independent” means that the release kinetics ofprostacyclin from the prostacyclin compound measured at 37° C.independently in buffer at pH 7.4 and in buffered 80% plasma at pH 7.4varies by no more than 50%, preferably by no more than 40%, morepreferably by no more than 30%, even more preferably by no more than 20%and most preferably by no more than 10%.

The term “enzyme-independent” means that the release of prostacyclinfrom the prostacyclin compound does not require the presence of enzymes.

Preferably, the prostacyclin compound of all above described aspects andembodiments of the present invention comprises a prostacyclin compoundor pharmaceutical composition for use which is in a depot, preferably apolymer gel, more preferably a hydrogel, e.g. a well hydrated polymermatrix.

In an alternatively preferred embodiment, the prostacyclin compound orpharmaceutical composition for use of all above described aspects andembodiment of the present invention is covalently linked to a compoundin the depot, preferably to the polymer gel, even more preferably thehydrogel, most preferably to the well hydrated polymer matrix.

Even more preferably, the pharmaceutical compositions or pharmaceuticalcomposition for use comprising a prostacyclin compound of the presentinvention are further characterized by being administered to a mammal,preferably a human, more preferably a human patient by injection, suchas subcutaneous or intramuscular injection.

Even more preferably, the prostacyclin compound comprised in apharmaceutical composition or pharmaceutical composition for use of allabove described aspects and embodiments of the present invention is aprodrug and which is further characterized in that after subcutaneous orintramuscular administration of said prostacyclin prodrug more than 50%of the administered prostacyclin dose is releasable within the bloodcompartment.

In addition, another aspect of the present invention is a pharmaceuticalcomposition or pharmaceutical composition for use comprising aprostacyclin prodrug which is characterized in that after subcutaneousor intramuscular administration of said prostacyclin prodrug more than50% of the administered prostacyclin dose is releasable within the bloodcompartment.

More preferably, the prostacyclin compound is a prodrug of prostacyclin,even more preferably a carrier-linked prostacyclin prodrug, even morepreferably a polymer carrier-linked prostacyclin prodrug, which meansthat the carrier moiety of the carrier-linked prostacyclin prodrugcomprises at least one polymer.

Preferably, the prostacyclin compound is a carrier-linked prostacyclinprodrug of formula (I):

Z¹X⁰-T)_(y)  (I),

wherein each T is independently a prostacyclin-comprising moiety,preferably the carrier-linked prostacyclin prodrug is a carrier-linkedtreprostinil prodrug of formula (I), wherein T is a moiety selected fromstructures (i) to (v):

-   -   wherein dashed lines indicating attachment to X⁰;

y is an integer ranging of from 1 to 64, preferably ranging from 1 to16, more preferably y selected from 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11,12, 13, 14, 15 and 16, even more preferably y is 8,

each X⁰ is independently (X^(0A))_(m1)-(X^(0B))_(m2);

m1; m2 are independently 0; or 1;

X^(0A) is T⁰;

X^(0B) is a branched or unbranched C₁₋₁₅ alkylene group which isunsubstituted or substituted with one or more R³, which are the same ordifferent;

R³ is halogen; C₁₋₆ alkyl; CN; C(O)R⁴; C(O)OR⁴; OR⁴; C(O)R⁴;C(O)N(R⁴R^(4a)); S(O)₂N(R⁴R^(4a)); S(O)N(R⁴R^(4a)); S(O)₂R⁴; S(O)R⁴;N(R⁴)S(O)₂N(R^(4a)R^(4b)); SR⁴; N(R⁴R^(4a)); NO₂; OC(O)R⁴;N(R⁴)C(O)R^(4a); N(R⁴)SO₂R^(4a); N(R⁴)S(O)R^(4a);N(R⁴)C(O)N(R^(4a)R^(4b)); N(R⁴)C(O)OR^(4a); OC(O)N(R⁴R^(4a)); or T⁰;

R⁴, R^(4a), R^(4b) are independently selected from the group consistingof H; T⁰; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, wherein C₁₋₆alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substituted withone or more R⁵, which are the same of different;

R⁵ is halogen; CN; C(O)R⁶; C(O)OR⁶; OR⁶; C(O)R⁶; C(O)N(R⁶R^(6a));S(O)₂N(R⁶R^(6a)); S(O)N(R⁶R^(6a)); S(O)₂R⁶; S(O)R⁶;N(R⁶)S(O)₂N(R^(6a)R^(6b)); SR⁶; N(R⁶R^(6a)); NO₂; OC(O)R⁶;N(R⁶)C(O)R^(6a); N(R⁶)SO₂R^(6a); N(R⁶)S(O)R^(6a);N(R⁶)C(O)N(R^(6a)R^(6b)); N(R⁶)C(O)OR^(6a); OC(O)N(R⁶R^(6a));

R⁶, R^(6a), R^(6b) are independently selected from the group consistingof H; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, wherein C₁₋₆ alkyl;C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substituted with one ormore halogen, which are the same of different;

T⁰ is phenyl; naphthyl; azulenyl; indenyl; indanyl; C₃₋₇ cycloalkyl; 3to 7 membered heterocyclyl; or 8 to 11 membered heterobicyclyl, whereinT⁰, is optionally substituted with one or more R⁷, which are the same ordifferent;

R⁷ is halogen; CN; COOR⁸; OR⁸; C(O)R⁸; C(O)N(R⁸R^(8a));S(O)₂N(R⁸R^(8a)); S(O)N(R⁸R^(8a)); S(O)₂R⁸; S(O)R⁸;N(R⁸)S(O)₂N(R^(8a)R^(8b)); SR⁸; N(R⁸R^(8a)); NO₂; OC(O)R⁸;N(R⁸)C(O)R^(8a); N(R⁸)S(O)₂R^(8a); N(R⁸)S(O)R^(8a); N(R⁸)C(O)OR^(8a);N(R⁸)C(O)N(R^(8a)R^(8b)); OC(O)N(R⁸R^(8a)); oxo (═O), where the ring isat least partially saturated; C₁₋₆ alkyl; C₂₋₆ alkenyl; or C₂₋₆ alkynyl,wherein C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionallysubstituted with one or more R⁹, which are the same or different;

R⁸, R^(8a), R^(8b) are independently selected from the group consistingof H; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, wherein C₁₋₆ alkyl;C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substituted with one ormore R¹⁰, which are the same of different;

R⁹, R¹⁰ are independently selected from the group consisting of halogen;CN; C(O)R¹¹; C(O)OR¹¹; OR¹¹; C(O)R¹¹; C(O)N(R¹¹R^(11a));S(O)₂N(R¹¹R^(11a)); S(O)N(R¹¹R^(11a)); S(O)₂R¹¹; S(O)R¹¹;N(R¹¹)S(O)₂N(R^(11a)R^(11b)); SR¹¹; N(R¹¹R^(11a)); NO₂; OC(O)R¹¹;N(R¹¹)C(O)R^(11a); N(R¹¹)SO₂R^(11a); N(R¹¹)S(O)R^(11a);N(R¹¹)C(O)N(R^(11a)R^(11b)); N(R¹¹)C(O)OR^(11a); and OC(O)N(R¹¹R^(11a));

R¹¹, R^(11a), R^(11b) are independently selected from the groupconsisting of H; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, whereinC₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substitutedwith one or more halogen, which are the same of different;

Z¹ is a carrier comprising a covalently bound polymer, preferably apharmaceutically acceptable polymer,

wherein the carrier is covalently attached to a moiety X⁰, provided thatone of m1, m2 is 1 and wherein the carrier is covalently attached to Tin case m1, m2=0,

or a pharmaceutically acceptable salt thereof.

It was surprisingly found that such carrier-linked treprostinil prodrugscan be used to obtain dosage forms of treprostinil which at leastpartially overcome the above mentioned shortcomings

Within the present invention the terms are used having the meaning asfollows.

In the present invention, the carrier-linked treprostinil prodrug or apharmaceutically acceptable salt thereof does not contain treprostinilin its free form or as a pharmaceutically acceptable salt thereof, butin bound form. Treprostinil is bound via one of its functional groups,e.g. via a hydroxyl or carboxyl, to the rest of the molecule and is aspart of a moiety T which is connected to a moiety X⁰ or—if m1 and m2 areboth 0—to a moiety Z¹ of formula (I). This means that the carrier-linkedtreprostinil prodrug according to the present invention containstreprostinil as a biologically active moiety. Due to the cleavage of thebiologically active moiety from the carrier-linked treprostinil prodrugwhen administered to a patient in need thereof, treprostinil is releasedeither in its free form or as a pharmaceutically acceptable saltthereof. In other words, the carrier-linked treprostinil prodrugcontains one or more moieties T, which moiety T is each substituted witha moiety X⁰ (provided that at least one of m1 and m2 is 1), which inturn is covalently bound to a carrier Z¹. Said carrier comprises acovalently bound polymer, preferably a pharmaceutically acceptablepolymer with a molecular weight of at least 500 Dalton.

In another preferred embodiment, the molecular weight of the polymer,preferably a pharmaceutically acceptable polymer is up to 160 kDa,preferably up to about 100 kDa, even more preferably up to about 50 kDa.

Preferably, a moiety X⁰ (provided that at least one of m1 and m2 is 1)and a moiety T are connected through a carbonate or ester linkage, mostpreferably a moiety X⁰ and a moiety T are connected through an esterlinkage.

Preferably, a moiety X⁰ is unsubstituted. More preferably, each moietyX⁰ is unsubstituted.

In one preferred embodiment, m1 is 0 and m2 is 1.

In another preferred embodiment, both m1 and m2 are 0.

Preferably, a sub structure X⁰—Z¹ is C(R¹R²)—CH₂—Z¹, wherein R¹, R² areindependently selected from the group consisting of H and C₁₋₄ alkyl,provided that at least one of R¹, R² is other than H; or (CH₂)_(n)—Z¹,wherein n is 2, 3, 4, 5, 6, 7 or 8.

Preferably, the carrier Z¹ is covalently attached to a moiety X⁰ via anamide group.

Preferably, R³ is halogen; CN; C(O)R⁴; C(O)OR⁴; OR⁴; C(O)R⁴;C(O)N(R⁴R^(4a)); S(O)₂N(R⁴R^(4a)); S(O)N(R⁴R^(4a)); S(O)₂R⁴; S(O)R⁴;N(R⁴)S(O)₂N(R^(4a)R^(4b)); SR⁴; N(R⁴R^(4a)); NO₂; OC(O)R⁴;N(R⁴)C(O)R^(4a); N(R⁴)SO₂R^(4a); N(R⁴)S(O)R^(4a);N(R⁴)C(O)N(R^(4a)R^(4b)); N(R⁴)C(O)OR^(4a); OC(O)N(R⁴R^(4a)); or T⁰.

Preferably, R⁴, R^(4a), R^(4b) are independently selected from the groupconsisting of H; T⁰; C₁₋₄ alkyl; C₂₋₄ alkenyl; and C₂₋₄ alkynyl, whereinC₁₋₄ alkyl; C₂₋₄ alkenyl; and C₂₋₄ alkynyl are optionally substitutedwith one or more R⁵, which are the same of different.

More preferably, the carrier-linked treprostinil prodrug is of formula(II):

-   -   wherein T, y and Z¹ are used as defined in formula (I), and    -   R^(a1) is selected from the group of unsubstituted alkyl;        substituted alkyl; unsubstituted phenyl; substituted phenyl;        unsubstituted naphthyl; substituted naphthyl; unsubstituted        indenyl; substituted indenyl; unsubstituted indanyl; substituted        indanyl; unsubstituted tetralinyl; substituted tetralinyl;        unsubstituted C₃₋₁₀ cycloalkyl; substituted C₃₋₁₀ cycloalkyl;        unsubstituted 4- to 7-membered heterocyclyl; substituted 4- to        7-membered heterocyclyl; unsubstituted 9- to 11-membered        heterobicyclyl; and substituted 9- to 11-membered        heterobicyclyl;    -   R^(a2) is selected from H, unsubstituted alkyl, and substituted        alkyl;    -   R^(a3) and R^(a4) are independently selected from the group        consisting of H, unsubstituted alkyl, and substituted alkyl;    -   n is 0 or 1;    -   optionally, R^(a1) and R^(a3) are joined together with the atoms        to which they are attached to form a ring A;    -   A is selected from the group consisting of C₃₋₁₀ cycloalkyl; 4-        to 7-membered aliphatic heterocyclyl; and 9- to 11-membered        aliphatic heterobicyclyl, wherein A is unsubstituted or        substituted;    -   Q is a spacer moiety;

Preferably, R^(a1) is C₁₋₆ alkyl or substituted C₁₋₆ alkyl, morepreferably C₁₋₄ alkyl or substituted C₁₋₄ alkyl.

More preferably, R^(a1) is selected from methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and benzyl.

Preferably, R^(a2) is H.

Preferably, R^(a3) is H, C₁₋₆ alkyl or substituted C₁₋₆ alkyl, morepreferably C₁₋₄ alkyl or substituted C₁₋₄ alkyl. More preferably, R^(a3)is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, and benzyl.

More preferably, R^(a3) is H.

Preferably, R^(a4) is selected from H, C₁₋₆ alkyl or substituted C₁₋₆alkyl, more preferably C₁₋₄ alkyl or substituted C₁₋₄ alkyl. Morepreferably, R^(a4) is selected from methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, t-butyl, and benzyl.

More preferably, R^(a4) is H.

In another preferred embodiment, R^(a1) and R^(a3) are joined togetherwith the atoms to which they are attached to form a ring A; wherein A isselected from the group consisting of cyclopropane, cyclobutane,cyclopentane, cyclohexane, cycloheptane.

Preferably, Q in formula (II) is selected from COOR^(a9); OR^(a9);C(O)R^(a9); C(O)N(R^(a9)R^(a9a)); S(O)₂N(R^(a9)R^(a9a));S(O)N(R^(a9)R^(a9a)); S(O)₂R^(a9); S(O)R^(a9);N(R^(a9))S(O)₂N(R^(a9a)R^(a9b)); SR^(a9); N(R^(a9)R^(a9a)); OC(O)R^(a9);N(R^(a9))C(O)R^(a9a); N(R^(a9))S(O)₂R^(a9a); N(R^(a9))S(O)R^(a9a);N(R^(a9))C(O)OR^(a9a); N(R^(a9))C(O)N(R^(a9a)R^(a9b));OC(O)N(R^(a9)R^(a9a)), W; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl,

wherein W, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R^(a10), which are the same or different,

and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of—W—, —C(O)O—; —O—; —C(O)—; —C(O)N(R^(a11))—; —S(O)₂N(R^(a11))—;—S(O)N(R^(a11))—; —S(O)₂—; —S(O)—; —N(R^(a11))S(O)₂N(R^(a11a))—; —S—;—N(R^(a11))—; —OC(O)R^(a11); —N(R^(a11))C(O)—; —N(R^(a11))S(O)₂—;—N(R^(a11))S(O)—; —N(R^(a11))C(O)O—; —N(R^(a11))C(O)N(R^(a11a))—; and—OC(O)N(R^(a11)R^(a11a));

R^(a9), R^(a9a), R^(a9b) are independently selected from the groupconsisting of H; W; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl,

wherein W, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R^(a10), which are the same or different,

and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting ofW, —C(O)O—; —O—; —C(O)—; —C(O)N(R^(a11))—; —S(O)₂N(R^(a11))—;—S(O)N(R^(a11))—; —S(O)₂—; —S(O)—; —N(R^(a11))S(O)₂N(R^(a11a))—; —S—;—N(R^(a11))—; —OC(O)R^(a11); —N(R^(a11))C(O)—; —N(R^(a11))S(O)₂—;—N(R^(a11))S(O)—; —N(R^(a11))C(O)O—; —N(R^(a11))C(O)N(R^(a11a))—; and—OC(O)N(R^(a11)R^(a11a));

W is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4- to 7-membered heterocyclyl; or9- to 11-membered heterobicyclyl, wherein W is optionally substitutedwith one or more R^(a10), which are the same or different;

R^(a10) is halogen; CN; oxo (═O); COOR^(a12); OR^(a12); C(O)R^(a12);C(O)N(R^(a12)R^(a12a)); S(O)₂N(R^(a12)R^(a12a)); S(O)N(R^(a12)R^(a12a));S(O)₂R^(a12); S(O)R^(a12); N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)); SR^(a12);N(R^(a12)R^(a12a)); NO₂; OC(O)R^(a12); N(R^(a12))C(O)R^(a12a);N(R^(a12))S(O)₂R^(a12a); N(R^(a12))S(O)R^(a12a);N(R^(a12))C(O)OR^(a12a); N(R^(a12))C(O)N(R^(a12a)R^(a12b));OC(O)N(R^(a12)R^(a12a)); or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different;

R^(a11), R^(a11a); R^(a12); R^(a12a); R^(a12b) are independentlyselected from the group consisting of H; and C₁₋₆ alkyl, wherein C₁₋₆alkyl is optionally substituted with one or more halogen, which are thesame or different.

In formula (II) the moiety X⁰ is of formula (IIa):

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        T and the unmarked dashed line indicates attachment to the rest        of the carrier-linked treprostinil prodrug; and    -   wherein Q, R^(a1), R^(a2), R^(a3), and R^(a4) are used as        defined in formula (II).

Preferably, X⁰ is selected from the following structures:

-   -   wherein dashed lines marked with an asterisk indicate attachment        to T and    -   unmarked dashed lines indicate attachment to the rest of the        carrier-linked treprostinil prodrug.

Preferably, all moieties T of the carrier-linked treprostinil prodrug offormula (I) have the same structure.

Preferably, all moieties T of formula (I) have the structure of formula(v).

Preferred sub-structures —X⁰-T of formula (I) are selected from thefollowing structures:

-   -   wherein the dashed lines indicate attachment to Z¹.

The carrier Z¹ comprises a covalently bound polymer, preferably apharmaceutically acceptable polymer.

Preferred polymers are selected from 2-methacryloyl-oxyethyl phosphoylcholins, hydrogels, PEG-based hydrogels, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyloxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, mannans, pectins, rhamnogalacturonans, starches,hydroxyalkyl starches, hydroxyethyl starches and othercarbohydrate-based polymers, xylans, and copolymers thereof.

Preferably, the carrier Z¹ comprises a poly(oxazoline) or a PEG-basedpolymer. Most preferably, the carrier Z¹ comprises a PEG-based polymer.

In one embodiment the carrier Z¹ may be a hydrogel (as one option for apolymer) which are known in the art. Suitable hydrogels are described inWO-A 2006/003014 or EP-A 1 625 856. If the carrier Z¹ is a hydrogel itis preferred that it is a PEG-based hydrogel as disclosed in WO-A2011/012715 which is incorporated by reference herewith.

Preferably, the carrier Z¹ is a water-soluble carrier.

In one embodiment the carrier Z¹ has the structure of formula (III):

wherein dashed lines indicate attachment to X⁰ and

wherein each of m, n, and p of formula (III) are independently aninteger ranging of from 5 to 500,

and wherein q of formula (III) ranges of from 2 to 32.

Preferably, q in formula (II) is an integer ranging of from 2 to 14 andmore preferably q of formula (II) is 6.

Preferably, each of m, n, and p in formula (III) independently range offrom 10 to 250, more preferably from 50 to 150. Preferably, m, n, and pin formula (III) are the same.

In an alternative embodiment the carrier Z¹ has the structure of formula(IV):

wherein dashed lines indicate attachment to X⁰, provided that one of m1,m2 is 1 and wherein the carrier is covalently attached to T in case m1,m2=0, and

wherein each of m, n, and p of formula (IV) are independently an integerranging of from 5 to 500 and wherein q of formula (IV) ranges of from 0to 14.

Preferably, q in formula (IV) is an integer ranging of from 2 to 6 andmore preferably q of formula (IV) is 2.

Preferably, each of m, n, and p in formula (IV) independently range offrom 10 to 250, more preferably from 50 to 150. Preferably, m, n, and pin formula (IV) are the same.

In another preferred embodiment Z¹ has the structure of formula (V):

Hyp¹ _(mx)-POL^(x)-Hyp²  (V),

-   -   wherein    -   POL^(x) is a polymeric moiety having a molecular weight ranging        from 0.5 kDa to 160 kDa,    -   Hyp¹ and Hyp² are independently a hyperbranched moiety, and    -   mx is 0 or 1.

The polymeric moiety POL^(x) has a molecular weight of from 0.5 kDa to160 kDa, preferably of from 2 kDa to 80 kDa and more preferably of from5 kDa to 40 kDa.

POL^(x) may be selected from the group of polymers consisting of, forexample, polypeptides, 2-methacryloyl-oxyethyl phosphoyl cholins,water-soluble hydrogels, water-soluble PEG-based hydrogels,water-soluble hyaluronic acid-based hydrogels, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyloxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

The polymeric moiety POL^(x) of formula (V) may comprise a linear orbranched polymer. Preferably, POL^(x) of formula (V) comprises, inparticular consists of a linear polymer.

In one preferred embodiment, POL^(x) of formula (V) comprises, inparticular consists of a PEG-based polymer or a poly(oxazoline)-basedpolymer, more preferably a linear PEG-based polymer. Even morepreferably, POL^(x) of formula (V) consists of a PEG-based linearpolymer.

If m in formula (V) is 0, it is preferred that POL^(x) of formula (V)comprises, preferably consists of a structure of the formulaX1-(OCH₂CH₂)_(p)—O—(CH₂)_(n)—X2-, wherein n is selected from 2, 3, or 4;p is an integer in the range of from 5 to 2000, preferably p is aninteger in the range of from 10 to 1000, more preferably p is an integerin the range of from 100 to 1000; and X2 is a functional groupcovalently linking POL^(x) and Hyp² of formula (V); and X1 is selectedfrom H, CH₃ and C₂H₅.

If m in formula (V) is 1, it is preferred that POL^(x) of formula (V)comprises, preferably consists of a structure of the formulaX3-(CH₂)_(n1)—(OCH₂CH₂)_(p)—O—(CH₂)_(n2)—X2-, wherein n1 and n2 areindependently selected from 2, 3, and 4; p is an integer in the range offrom 5 to 2000, preferably p is an integer in the range of from 10 to1000, more preferably p is an integer in the range of from 100 to 1000;and X2 and X3 are functional groups covalently linking POL^(x) to Hyp¹and Hyp² of formula (V), respectively.

In a preferred embodiment mx in formula (V) is 0.

In another preferred embodiment, POL^(x) of formula (V) is a polypeptide(or protein), in particular a non-immunogenic polypeptide as describedbelow.

Preferably, the polymeric moiety POL^(x) of formula (V) is a polypeptidewhich comprises at least about 100 amino acid residues, in particularwhich consists of at least about 100 amino acid residues. In a preferredembodiment, amino acids selected from alanine, serine and/or prolineresidues are present, in particular are mainly present, and whichpolypeptide moiety preferably has a random coil conformation atphysiological conditions. It is understood that such a polypeptidemoiety POL^(x) of formula (V) may transiently or temporarily not form arandom coil, for example when present in a lyophilisate or driedcomposition.

A polypeptide moiety POL^(x) of formula (V) may have a random coilconformation with an amino acid sequence consisting of maximally about1000 amino acid residues, preferably of maximally about 900 amino acidresidues, more preferably of maximally about 800 amino acid residues,even more preferably of maximally about 700 amino acid residues,particularly preferably of maximally about 600 amino acid residues.Thus, the amino acid sequence forming random coil conformation mayconsist of maximally about 500 amino acid residues or of maximally about450 amino acid residues.

It is also envisaged herein that the amino acid sequence forming randomcoil conformation may consist of maximally about 1200 and up to about1500 amino acid residues. Accordingly, the amino acid sequence formingrandom coil conformation may consist of about 100 to about 1500 aminoacid residues.

In particular embodiments said amino acid sequence forming random coilconformation consists of about 100 to 1000 amino acid residues ascharacterized herein, i.e. comprising alanine, serine and/or proline asmain or unique residues as defined below.

In a preferred embodiment, a polypeptide moiety POL^(x) of formula (V)consists mainly of one, two or three of the amino acid residues alanine,serine and proline, whereby proline residues represent preferably about4% to about 40% of the polypeptide moiety POL^(x) of formula (V). Thealanine and serine residues comprise the remaining at least 60% to 96%of the polypeptide moiety POL^(x) of formula (V). However, as will bedetailed herein below said polypeptide moiety POL^(x) of formula (V) mayalso comprise further amino acids differing from alanine, serine, andproline, i.e. as minor constituents.

The term “minor constituent” as used herein means that maximally 10%(i.e. maximally 10 of 100 amino acids) may be different from alanine,serine and proline, preferably maximally 8% (i.e. maximally 8 of 100amino acids) may be different than alanine, serine and proline, morepreferably maximally 6% (i.e. maximally 6 of 100 amino acids) may bedifferent from alanine, serine and proline, even more preferablymaximally 5% (i.e. maximally 5 of 100 amino acids) may be different fromalanine, serine and proline, particularly preferably maximally 4% (i.e.maximally 4 of 100 amino acids) may be different from alanine, serineand proline, more particularly preferably maximally 3% (i.e. maximally 3of 100 amino acids) may be different from alanine, serine and proline,even more particularly preferably maximally 2% (i.e. maximally 2 of 100amino acids) may be different from alanine, serine and proline and mostpreferably maximally 1% (i.e. maximally 1 of 100 of the amino acids) maybe different from alanine, serine and proline. Said amino acidsdifferent from alanine, serine and proline may be selected from thegroup consisting of different from alanine, serine and proline may beselected from the group of natural or proteinogenic amino-acidscomprising Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Thr, Trp, Tyr, Val, selenocystein, selenomethionin, andhydroxyproline. Minor constituents may also be selected fromnon-naturally occurring amino acids.

The term “at least about 100/150/200/250/300/300/350 (etc) amino acidresidues” is not limited to the concise number of amino acid residuesbut also comprises amino acid stretches that comprise an additional 10%to 20% or comprise 10% to 20% less residues. For example “at least about100 amino acid residues” may also encompass 80 to 100 and about 100 to120 amino acid residues without deferring from the gist of the presentinvention.

In one embodiment, the polypeptide moiety POL^(x) of formula (V)comprises a plurality of polymer cassettes wherein said polymercassettes consist of one, two or three of the amino acids selected fromAla, Ser, and Pro and wherein no more than 6 consecutive amino acidresidues are identical and wherein said proline residues constitute morethan 4% and less than 40% of the amino acids of said polypeptide moietyPOL^(x) of formula (V).

A polypeptide moiety POL^(x) of formula (V) may comprise a plurality, inparticular 2, 3, 4, 5 or more of identical polymer cassettes or aplurality of non-identical polymer cassettes. Non-limiting examples ofpolymer cassettes consisting of Ala, Ser and Pro residues are providedherein below; see SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13 and SEQ ID NO:14 or peptide fragments or multimers of thesesequences. A polymer cassette may consist of at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more amino acid residues, wherein each polymercassette comprises (an) Ala, Ser, and Pro residue(s).

In one embodiment, the polymer cassette according to the presentinvention does not comprise more than 100 amino acid residues.Preferably, a polymer cassette as defined herein comprises more thanabout 4%, preferably more than about 5%, even more preferably more thanabout 6%, particularly preferably more than about 8%, more particularlypreferably more than about 10%, even more particularly preferably morethan about 15% and most preferably more than about 20% proline residues.Such polymer cassette as defined herein preferably comprises less thanabout 40% or less than about 35% proline residues.

In one preferred embodiment the polypeptide moiety POL^(x) of formula(V) comprises, in particular consists of formula (a):

Ser_(x)[Ala_(y)Ser_(z)]_(n)  (a),

-   -   which formula further comprises proline residues as defined        herein and wherein    -   x is independently selected from integer 0 to 6,    -   each y is independently selected from integer ranging of from 1        to 6,    -   each z is independently selected from integer ranging of from 1        to 6.    -   n is any integer so that a polypeptide moiety POL^(x) of        formula (V) consists of at least about 100 amino acid residues,        and in particular of at least about 100 to about 3000 amino acid        residues, preferably to about 2000 and more preferably to about        1000 amino acid residues.

In another preferred embodiment, a polypeptide moiety POL^(x) of formula(V) comprises no more than 5 identical consecutive amino acid residues,more preferably no more than 4 identical consecutive amino acid residuesand most preferably no more than 3 identical consecutive amino acidresidues.

As already indicated herein above, a polypeptide moiety POL^(x) offormula (V) comprises in one embodiment proline residues, wherein saidproline residues constitute more than about 4%, preferably more thanabout 5%, even more preferably more than about 6%, particularlypreferably more than about 8%, more particularly preferably more thanabout 10%, even more particularly preferably more than about 15% andmost preferably more than about 20% of the amino acids of POL^(x) offormula (V).

In another preferred embodiment, a polypeptide moiety POL^(x) of formula(V) comprises more than about 4% but less than about 50%, preferablymore than about 10% but less than about 50% and most preferably morethan about 20% but less than about 50% alanine residues of the aminoacids constituting the polypeptide moiety POL^(x) of formula (V).

In a further preferred embodiment, a polypeptide moiety POL^(x) offormula (V) comprises more than about 4% and less than about 50%,preferably more than about 10% but less than about 50% and mostpreferably more than about 20% but less than about 50% serine residuesof the amino acids constituting the polypeptide moiety POL^(x) offormula (V).

Preferably, a polypeptide moiety POL^(x) of formula (V) comprises about35% proline residues, about 50% alanine residues and about 15% serineresidues of the amino acids constituting the polypeptide moiety POL^(x)of formula (V). Alternatively, a polypeptide moiety POL^(x) of formula(V) may comprise about 35% proline residues, about 15% alanine residuesand about 50% serine residues of the amino acids constituting thepolypeptide moiety POL^(x) of formula (V).

Preferably, a polypeptide moiety POL^(x) of formula (V) comprises one ormore of the following alanine-serine polymer cassettes:

SEQ ID NO: 1 AAAASSASSASSSSSAAASA SEQ ID NO: 2 AASAAASSAAASAAAASASSSEQ ID NO: 3 ASASASASASASSAASAASA SEQ ID NO: 4 SAASSSASSSSAASSASAAASEQ ID NO: 5 SSSSAASAASAAAAASSSAS SEQ ID NO: 6 SSASSSAASSSASSSSASAASEQ ID NO: 7 SASASASASASAASSASSAS SEQ ID NO: 8 ASSAAASAAAASSAASASSS

The multimers of these alanine-serine polymer cassettes may form randomcoil conformation in case the resulting amino acid sequence furthercomprises proline residues as defined herein above.

In a preferred embodiment, a polypeptide moiety POL^(x) of formula (V)comprises one or more of the following polymer cassettes:

SEQ ID NO: 9 ASPAAPAPASPAAPAPSAPA SEQ ID NO: 10 AAPASPAPAAPSAPAPAAPSSEQ ID No: 11 APSSPSPSAPSSPSPASPSS SEQ ID NO: 15 SAPSSPSPSAPSSPSPASPS

SEQ ID NO:15 corresponds to the herein provided SEQ ID No:11 in acircularly permuted form, wherein the last serine was removed andanother serine was appended as starting amino acid. As a consequence,multimers of this modified sequence possess essentially the sameinternal repeating unit as multimers of the non-modified sequence,except for the very first and the very last residue. Accordingly, SEQ IDNO:15 may be considered as an example of a further polymer cassette fora polypeptide moiety POL^(x) of formula (V). It is clear for the personskilled in the art that also other polymer cassettes and (shorter)peptide fragments or circularly permuted versions of the herein providedamino acid polymers may be used as polymer cassettes for a polypeptidemoiety POL^(x) of formula (V).

Yet, even further and illustrative amino acid polymers forming randomcoil conformation may comprise amino acid sequences that may be selectedfrom the group consisting of the following sequences:

SEQ ID NO: 12 SSPSAPSPSSPASPSPSSPA SEQ ID NO: 13AASPAAPSAPPAAASPAAPSAPPA SEQ ID NO: 14 ASAAAPAAASAAASAPSAAA

Therefore, preferred polymer cassettes for a polypeptide moiety POL^(x)of formula (V) are selected from the following sequences:

(SEQ ID NO: 9) ASPAAPAPASPAAPAPSAPA, (SEQ ID NO: 10)AAPASPAPAAPSAPAPAAPS, (SEQ ID NO: 11) APSSPSPSAPSSPSPASPSS,(SEQ ID NO: 12) SSPSAPSPSSPASPSPSSPA, (SEQ ID NO: 13)AASPAAPSAPPAAASPAAPSAPPA, and (SEQ ID NO: 14) ASAAAPAAASAAASAPSAAA;

-   -   or circular permuted versions or (a) multimer(s) of these        sequences as a whole or parts of these sequences.

Again, also (a) peptide fragment(s) or (a) multimer(s) or circularlypermuted versions of these sequences and the sequences provided hereinabove may be employed in context of the present invention as polymercassettes for a polypeptide moiety POL^(x) of formula (V). The personskilled in the art is readily in a position to generate further aminoacid polymer cassettes that form random coil conformation underphysiological conditions and are constituted of mainly alanine, serine,and proline as defined herein. Such other and further examples of randomcoil conformation forming amino acid polymer cassettes to be used for apolypeptide moiety POL^(x) of formula (V) may, inter alia, comprisecombinations and/or peptide fragments or circularly permuted versions ofthe specific polymer cassettes shown above.

Accordingly, the exemplified polymer cassettes may also provide forindividual peptide fragments which may be newly combined to form furtherpolymer cassettes.

In accordance with the above, a polypeptide moiety POL^(x) of formula(V) may comprise a multimer of sequences consisting of either one of theamino acid sequences with SEQ ID NO:9, 10, 11, 12, 13 or 14 as disclosedherein above or may comprise a multimer of sequences consisting of morethan one of amino acid sequences SEQ ID NOs:9, 10, 11, 12, 13 and 14.Furthermore, it is envisaged that also peptide fragments or circularlypermuted versions of these exemplified sequences may be used to build upfurther polymer cassettes of a polypeptide moiety POL^(x) of formula(V).

In another embodiment, a polypeptide moiety POL^(x) of formula (V) maycomprise a multimer of sequences consisting of a (circular) permutationof the amino acid sequence selected from the group consisting of SEQ IDNO:9, 10, 11, 12, 13, 14, 15 or (a) multimers(s) of these (circular)permutated sequences.

In yet another embodiment, a polypeptide moiety POL^(x) of formula (V)may comprise a multimer consisting of a peptide fragment/part of theamino acid sequence selected from the group consisting of SEQ ID NOs: 9,10, 12, 13, 14, 15 or (a) multimers(s) of these exemplified polymercassettes.

Peptide fragments of these sequences to be employed for the generationof a polypeptide moiety POL^(x) of formula (V) may consist of at least3, preferably of at least 4, more preferably of at least 5, even morepreferably of at least 6, still more preferably of at least 8,particularly preferably of at least 10, more particularly preferably ofat least 12, even more particularly preferably of at least 14,preferably of at least 6, still more preferably of at least 8,particularly preferably of at least 10, more particularly preferably ofat least 12, even more particularly preferably of at least 14, even moreparticularly preferably of at least 16, and most preferably of at least18 consecutive amino acids of the amino acid sequence selected from thegroup consisting of said SEQ ID NOs: 9, 10, 11, 12, 13 and 14.

For example, individual peptide fragments of the inventive polymercassettes may be combined to further individual polymer cassettes aslong as the above-identified rules for the overall distribution andamount of alanine, serine and proline are respected. Again, thesepolymer cassettes may also comprise further amino acid residues, howeveronly as minimal or minor constituents, i.e. maximally 10%, preferablymaximally 2% of the individual polymer cassette. POL^(x) of formula (V)moieties comprising polymer cassettes consist, in one embodiment of thepresent invention, of at least about 100 amino acid residues. Individualpolymer cassettes may be combined in order to form longer random coilforming amino acid polymers, whereby a maximal length of a polypeptidemoiety POL^(x) of formula (V) is about 3000 amino acids.

Preferably, POL^(x) of formula (V) is covalently linked to Hyp¹ and Hyp²of formula (V), in particular by a permanent linkage, more preferably bya permanent amide linkage.

In the carrier-linked treprostinil prodrugs of the present inventionfunctional groups of Hyp¹ and Hyp² of formula (V) are connected to amoiety X⁰ of formula (I).

The hyperbranched moieties Hyp¹ and Hyp² of formula (V) are eachindependently selected from the group comprising, in particularconsisting of, in bound form glycerol, pentaerythritol,dipentaerythritol, tripentaerythritol, hexaglycerine, sucrose, sorbitol,fructose, mannitol, glucose, cellulose, amyloses, starches, hydroxyalkylstarches, polyvinylalcohols, dextranes, hyualuronans, dilysine,trilysine, tetralysine, pentalysine, hexylysine, heptalysine,octalysine, nonalysine, decalysine, undecalysine, dodecalysine,tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine,heptadecalysine, octadecalysine, nonadecalysine, triornithine,tetraornithine, pentaornithine, hexaornithine, heptaornithine,octaornithine, nonaornithine, decaornithine, undecaornithine,dodecaornithine, tridecaornithine, tetradecaornithine,pentadecaornithine, hexadecaornithine, heptadecaornithine,octadecaornithine, nonadecaornithine, tridiaminobutyric acid,tetradiaminobutyric acid, pentadiaminobutyric acid, hexadiaminobutyricacid, heptadiaminobutyric acid, octadiaminobutyric acid,nonadiaminobutyric acid, decadiaminobutyric acid, undecadiaminobutyricacid, dodecadiaminobutyric acid, tridecadiaminobutyric acid,tetradecadiaminobutyric acid, pentadecadiaminobutyric acid,hexadecadiaminobutyric acid, heptadecadiaminobutyric acid,octadecadiaminobutyric acid, nonadecadiaminobutyric acid, di(glutamicacid), tri(glutamic acid), tetra(glutamic acid), penta(glutamic acid),hexa(glutamic acid), hepta(glutamic acid), octa(glutamic acid),nona(glutamic acid), deca(glutamic acid), undeca(glutamic acid),dodeca(glutamic acid), trideca(glutamic acid), tetradeca(glutamic acid),pentadeca(glutamic acid), hexadeca(glutamic acid), heptadeca(glutamicacid), octadeca(glutamic acid), nonadeca(glutamic acid), di(asparticacid), tri(aspartic acid), tetra(aspartic acid), penta(aspartic acid),hexa(aspartic acid), hepta(aspartic acid), octa(aspartic acid),nona(aspartic acid), deca(aspartic acid), undeca(aspartic acid),dodeca(aspartic acid), trideca(aspartic acid), tetradeca(aspartic acid),pentadeca(aspartic acid), hexadeca(aspartic acid), heptadeca(asparticacid), octadeca(aspartic acid), nonadeca(aspartic acid),polyethyleneimines, and low-molecular weight PEI.

In a preferred embodiment, the hyperbranched moieties Hyp¹ and Hyp² offormula (V) are each independently selected from the group comprising,in particular consisting of, in bound form dilysine, trilysine,tetralysine, pentalysine, hexylysine, heptalysine, octalysine,nonalysine, decalysine, undecalysine, dodecalysine, tridecalysine,tetradecalysine, pentadecalysine, hexadecalysine, heptadecalysine,octadecalysine, nonadecalysine, triornithine, tetraornithine,pentaornithine, hexaornithine, heptaornithine, octaornithine,nonaornithine, decaornithine, undecaornithine, dodecaornithine,tridecaornithine, tetradecaornithine, pentadecaornithine,hexadecaornithine, heptadecaornithine, octadecaornithine,nonadecaornithine, tridiaminobutyric acid, tetradiaminobutyric acid,pentadiaminobutyric acid, hexadiaminobutyric acid, heptadiaminobutyricacid, octadiaminobutyric acid, nonadiaminobutyric acid,decadiaminobutyric acid, undecadiaminobutyric acid, dodecadiaminobutyricacid, tridecadiaminobutyric acid, tetradecadiaminobutyric acid,pentadecadiaminobutyric acid, hexadecadiaminobutyric acid,heptadecadiaminobutyric acid, octadecadiaminobutyric acid,nonadecadiaminobutyric acid, di(glutamic acid), tri(glutamic acid),tetra(glutamic acid), penta(glutamic acid), hexa(glutamic acid),hepta(glutamic acid), octa(glutamic acid), nona(glutamic acid),deca(glutamic acid), undeca(glutamic acid), dodeca(glutamic acid),trideca(glutamic acid), tetradeca(glutamic acid), pentadeca(glutamicacid), hexadeca(glutamic acid), heptadeca(glutamic acid),octadeca(glutamic acid), nonadeca(glutamic acid), di(aspartic acid),tri(aspartic acid), tetra(aspartic acid), penta(aspartic acid),hexa(aspartic acid), hepta(aspartic acid), octa(aspartic acid),nona(aspartic acid), deca(aspartic acid), undeca(aspartic acid),dodeca(aspartic acid), trideca(aspartic acid), tetradeca(aspartic acid),pentadeca(aspartic acid), hexadeca(aspartic acid), heptadeca(asparticacid), octadeca(aspartic acid), nonadeca(aspartic acid),polyethyleneimines, and low-molecular weight PEI.

More preferably, the hyperbranched moieties Hyp¹ and Hyp² of formula (V)are independently selected from the group comprising, more preferablyconsisting of, in bound form, trilysine, tetralysine, pentalysine,hexylysine, heptalysine, octalysine, nonalysine, decalysine,undecalysine, dodecalysine, tridecalysine, tetradecalysine,pentadecalysine, hexadecalysine, and heptadecalysine, even morepreferably Hyp¹ and Hyp² are independently comprising, preferablyconsisting of, in bound form, trilysine, heptalysine or pentadecalysine.

More preferably, Hyp¹ and Hyp² of formula (V) are independently selectedfrom any one of the following structures:

-   -   wherein    -   dashed lines marked with an asterisk indicate attachment to        POL^(x) of formula (V),    -   unmarked dashed lines indicate attachment to X⁰, and    -   qx is an integer of from 0 to 15, preferably 3 to 7, and even        more preferably 6.

Preferably, Hyp¹ and Hyp² of formula (V) are each a heptalysinyl group,in particular Hyp¹ and Hyp² of formula (V) each have the structure offormula (ii-x) above.

Preferably, Hyp¹ and Hyp² of formula (V) have the same structure.

Functional groups of Hyp¹ and Hyp² of formula (V) serve as attachmentpoints for direct linkage of Hyp¹ and Hyp² of formula (V) to X⁰.Remaining functional groups which are not connected to X⁰ may,independently of each other, be capped with suitable capping reagents ormay optionally be connected to at least one targeting moiety, inparticular through permanent linkages

Therefore, in the water-soluble carrier-linked prodrugs of the presentinvention the hyperlinked moieties Hyp¹ and Hyp² of formula (V) areconnected to POL^(x) of formula (V) and functional groups of Hyp¹ andHyp² of formula (V) are connected to X⁰ of formula (I), permanentlinkages, targeting moieties and/or capping groups.

In a preferred embodiment, all functional groups of the hyperbranchedmoieties Hyp¹ and Hyp² of formula (V) are connected to X⁰ of formula(I).

Preferably, the hyperbranched moieties Hyp¹ and Hyp² of formula (V) haveindependently a molecular weight in the range of from 0.1 kDa to 4 kDa,more preferably 0.4 kDa to 2 kDa. Preferably, the hyperbranched moietiesHyp¹ and Hyp² of formula (V) have each independently at least 3branchings and are each independently conjugated to at least 4 X⁰,permanent linkages, and/or capping groups and each independently have atmost 63 branchings and are each independently at most conjugated to 64X⁰, permanent linkages, and/or capping groups. It is preferred that thehyperbranched moieties Hyp¹ and Hyp² of formula (V) have eachindependently at least 7 branchings and are each independentlyconjugated to at least 8 X⁰, permanent linkages, and/or capping groupsand have each independently at most 31 branchings and are eachindependently at most conjugated to 32 X⁰, permanent linkages, and/orcapping groups.

Preferably, the hyperbranched moieties Hyp¹ and Hyp² of formula (V) areeach independently a hyperbranched polypeptide. Preferably, suchhyperbranched polypeptide comprises lysine in bound form. Preferably,each hyperbranched moiety Hyp¹ and Hyp² of formula (V) independentlyhave a molecular weight in the range of from 0.1 kDa to 4 kDa, inparticular 0.4 kDa to 2 kDa.

Preferably, mx is 0 and POL-Hyp²- of formula (V) is selected from thefollowing structures:

-   -   wherein    -   dashed lines indicate attachment to X⁰, provided that one of m1,        m2 is 1 and wherein the carrier is covalently attached to T in        case m1, m2=0,    -   px is an integer of from 5 to 2000, preferably 10 to 1000, in        particular 100 to 1000, and    -   qx is an integer of from 0 to 15, preferably 3 to 7, more        preferably, qx is 6.

In another preferred embodiment Z¹ of formula (I) has the structure offormula (VI):

BA-Hyp^(y))_(n)  (VI),

-   -   wherein    -   B is a branching core,    -   each A is independently a poly(ethylene glycol)-based polymeric        chain,    -   each Hyp^(y) is independently a branched moiety, and    -   n is an integer of from 3 to 32;

In a preferred embodiment, the branching core B of formula (VI)comprises, preferably consists of a moiety selected from:

-   -   a polyalcohol comprising at least 2 hydroxyl groups (preferably        further comprising a functional group, which is preferably an        additional amino group or a carboxylic acid group, more        preferably an additional carboxylic acid group),    -   preferably B is selected from glycerol, pentaerythritol,        dipentaerythritol, tripentaerythritol, hexaglycerine, sucrose,        sorbitol, fructose, mannitol, glucose, cellulose, amyloses,        starches, hydroxyalkyl starches, polyvinylalcohols, dextranes,        and hyualuronans,    -   or a polyamine comprising at least 2 amine groups (preferably        further comprising a functional group, which is preferably an        additional hydroxyl group or a carboxylic acid group, more        preferably a carboxylic acid group),    -   preferably selected from ornithine, diornithine, triornithine,        tetraornithine, pentaornithine, hexaornithine, heptaornithine,        octaornithine, nonaornithine, decaornithine, undecaornithine,        dodecaornithine, tridecaornithine, tetradecaornithine,        pentadecaornithine, hexadecaornithine, heptadecaornithine,        octadecaornithine, nonadecaornithine, diaminobutyric acid,        di(diaminobutyric acid), tri(diaminobutyric acid),        tetra(diaminobutyric acid), penta(diaminobutyric acid),        hexa(diaminobutyric acid), hepta(diaminobutyric acid),        octa(diaminobutyric acid), nona(diaminobutyric acid),        deca(diaminobutyric acid), undeca(diaminobutyric acid),        dodeca(diaminobutyric acid), trideca(diaminobutyric acid),        tetradeca(diaminobutyric acid), pentadeca(diaminobutyric acid),        hexadeca(diaminobutyric acid), heptadeca(diaminobutyric acid),        octadeca(diaminobutyric acid), nonadeca(diaminobutyric acid),        lysine, dilysine, trilysine, tetralysine, pentalysine,        hexylysine, heptalysine, octalysine, nonalysine, decalysine,        undecalysine, dodecalysine, tridecalysine, tetradecalysine,        pentadecalysine, hexadecalysine, heptadecalysine,        octadecalysine, nonadecalysine, oligolysines,        polyethyleneimines, and polyvinylamines;    -   wherein the polyalcohol or polyamine is in bound form.

In a preferred embodiment, the branching core B of formula (VI)comprises, preferably consists of pentaerithritol.

Preferably, a poly(ethylene glycol)-based polymeric chain A connected tothe branching core B of formula (VI) consists of a linear PEG chain, ofwhich one terminus is connected to B of formula (VI) and the otherterminus is connected to Hyp^(y) of formula (VI). It is understood thata PEG-based chain A of formula (VI) may optionally be terminated in caseof a branched PEG chain and/or may optionally be interrupted in case ofa branched or linear PEG chain by alkyl or aryl groups and mayoptionally be substituted with heteroatoms and/or functional groups.

Each sub-structure A-Hyp^(y) of formula (VI) extending from thebranching core B of formula (VI) may be independently of each other thesame or different sub-structures A-Hyp^(y). In a preferred embodiment,the all sub-structures A-Hyp^(y) of formula (VI) are the same.

Each A and each Hyp^(y) of formula (VI) may be independently selectedfrom the other moieties A and Hyp^(y). Preferably, all sub-structuresA-Hyp^(y) connected to B of formula (VI) have an identical structure.

Preferably, the PEG-based polymeric chains A of formula (VI) areconnected to B through permanent linkages.

n of formula (VI) is an integer from 3 to 32. Preferably, n is aninteger from 3 to 16, more preferably n is an integer from 4 to 8 andmost preferably n is 4.

In a preferred embodiment n of formula (VI) is 4 and m is 2.

In one embodiment, a PEG-based polymeric chain A of formula (VI) isselected from a linear or branched PEG-based polymeric chain.Preferably, A is a linear PEG-based polymeric chain.

Preferably, each A of formula (VI) is independently selected from theformula

—X3-(CH₂)_(n1)—(OCH₂CH₂)_(p)—O—(CH₂)_(n2)—X2-,

-   -   wherein    -   n1 and n2 are independently selected from 1, 2, 3, and 4,        preferably from 1, 2, and 3;    -   p is an integer in the range of from 5 to 2000, preferably p is        an integer in the range of from 10 to 1000, more preferably p is        an integer in the range of from 100 to 1000; and    -   X3 and X2 are independently functional groups covalently linked        to B or Hyp^(y), respectively.

Preferably, a linkage between a moiety A and a moiety Hyp^(y) of formula(VI) is a permanent linkage, more preferably a permanent linkagecomprising a linkage group comprising, in particular consisting of agroup selected from amine groups, amide groups, carbamate groups,thioether groups, ether groups, and most preferably a permanent linkagebetween a moiety A and a moiety Hyp^(y) of formula (VI) is an amidelinkage.

In a preferred embodiment, a sub-structure B-(A)_(n) of formula (VI) isa multi-arm PEG derivative as, for instance, detailed in the productslist of JenKem Technology, USA (accessed by download fromhttp://jenkemusa.net/pegproducts2.aspx on Mar. 8, 2011), such as a4-arm-PEG derivative, in particular comprising a pentaerythritol core,an 8-arm-PEG derivative comprising a hexaglycerin core, and an 8-arm-PEGderivative comprising a tripentaerythritol core. Most preferred aresub-structures B-(A)_(n) of formula (VI) comprising, in particularconsisting of, moieties selected from:

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 400 to 2000;

a 4-arm PEG Carboxyl comprising a pentaerythritol core:

with n ranging from 400 to 2000;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 400 to 2000 and

R=hexaglycerin core structure;

an 8-arm PEG Carboxyl comprising a hexaglycerin core:

with n ranging from 400 to 2000 and

R=hexaglycerin core structure;

an 8-arm PEG Amine comprising a tripentaerythritol core:

with n ranging from 400 to 2000

and R=tripentaerythritol core structure;

and an 8-arm PEG Carboxyl comprising a tripentaerythritol core:

with n ranging from 400 to 2000 and

R=tripentaerythritol core structure;

each in bound form.

In a preferred embodiment, the molecular weight of a sub-structureB-(A)_(n) of formula (VI) ranges from 1 kDa to 80 kDa, more preferably 1kDa to 40 kDa and even more preferably 10 kDa to 40 kDa. It isunderstood that the terminal amine groups or carboxyl groups,respectively, are used for conjugation to a moiety Hyp^(y) of formula(VI).

Functional groups of a moiety Hyp^(y) of formula (VI) are connected tomoieties X⁰ of formula (I).

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) is connectedto a moiety X⁰ of formula (I) through a functional group selected fromamide groups, carbamate groups, ester groups, ether groups, aminegroups, thioether groups. Preferably, a moiety Hyp^(y) of formula (VI)is connected to a moiety X⁰ of formula (I) through amide groups,thioether groups and/or ether groups, even more preferably through amidegroups.

Optionally, functional groups of a moiety Hyp^(y) of formula (VI) whichare not connected to a moiety X⁰ of formula (I) may be capped withsuitable capping reagents and/or may optionally be connected to at leastone targeting moiety, in particular through permanent linkages.Therefore, a moiety Hyp^(y) of formula (VI) may be connected to a moietyX⁰ of formula (I), capping moieties and/or targeting moieties.Preferably, functional groups of a moiety Hyp^(y) of formula (VI) areconnected to a moiety X⁰ of formula (I) and are not connected to cappingmoieties and/or targeting moieties. Targeting moieties, if present, maybe conjugated to a moiety Hyp^(y) of formula (VI) either directly orindirectly through spacer moieties.

Examples of suitable capping moieties are linear, branched or cyclicC₁₋₈ alkyl groups.

In one embodiment, each branched moiety Hyp^(y) of formula (VI) isdirectly or indirectly connected to at least two moieties X⁰ of formula(I). More preferably, each branched moiety Hyp^(y) of formula (VI) isdirectly or indirectly connected to at least three moieties X⁰ offormula (I). Most preferably, each branched moiety Hyp^(y) of formula(VI) is directly or indirectly connected to at least four moieties X⁰ offormula (I).

The branched moiety Hyp^(y) of formula (VI) comprises, preferablyconsists of a moiety in bound form selected from:

-   -   a polyalcohol in bound form comprising at least 2 hydroxyl        groups (preferably further comprising a functional group, which        is preferably an additional hydroxyl group or a carboxylic acid        group, more preferably an additional hydroxyl group),    -   preferably selected from glycerol, pentaerythritol,        dipentaerythritol, tripentaerythritol, hexaglycerine, sucrose,        sorbitol, fructose, mannitol, glucose, cellulose, amyloses,        starches, hydroxyalkyl starches, polyvinylalcohols, dextranes,        and hyualuronans,    -   or a polyamine in bound form comprising at least 2 amine groups        (preferably further comprising a functional group, which is        preferably an additional amine group or a carboxylic acid group,        more preferably a carboxylic acid group),    -   preferably selected from ornithine, diornithine, triornithine,        tetraornithine, pentaornithine, hexaornithine, heptaornithine,        octaornithine, nonaornithine, decaornithine, undecaornithine,        dodecaornithine, tridecaornithine, tetradecaornithine,        pentadecaornithine, hexadecaornithine, heptadecaornithine,        octadecaornithine, nonadecaornithine, diaminobutyric acid,        di(diaminobutyric acid), tri(diaminobutyric acid),        tetra(diaminobutyric acid), penta(diaminobutyric acid),        hexa(diaminobutyric acid), hepta(diaminobutyric acid),        octa(diaminobutyric acid), nona(diaminobutyric acid),        deca(diaminobutyric acid), undeca(diaminobutyric acid),        dodeca(diaminobutyric acid), trideca(diaminobutyric acid),        tetradeca(diaminobutyric acid), pentadeca(diaminobutyric acid),        hexadeca(diaminobutyric acid), heptadeca(diaminobutyric acid),        octadeca(diaminobutyric acid), nonadeca(diaminobutyric acid),        lysine, dilysine, trilysine, tetralysine, pentalysine,        hexylysine, heptalysine, octalysine, nonalysine, decalysine,        undecalysine, dodecalysine, tridecalysine, tetradecalysine,        pentadecalysine, hexadecalysine, heptadecalysine,        octadecalysine, nonadecalysine, oligolysines, triornithine,        tetraornithine, pentaornithine, hexaornithine, heptaornithine,        octaornithine, nonaornithine, decaornithine, undecaornithine,        dodecaornithine, tridecaornithine, tetradecaornithine,        pentadecaornithine, hexadecaornithine, heptadecaornithine,        octadecaornithine, nonadecaornithine, tridiaminobutyric acid,        tetradiaminobutyric acid, pentadiaminobutyric acid,        hexadiaminobutyric acid, heptadiaminobutyric acid,        octadiaminobutyric acid, nonadiaminobutyric acid,        decadiaminobutyric acid, undecadiaminobutyric acid,        dodecadiaminobutyric acid, tridecadiaminobutyric acid,        tetradecadiaminobutyric acid, pentadecadiaminobutyric acid,        hexadecadiaminobutyric acid, heptadecadiaminobutyric acid,        octadecadiaminobutyric acid, nonadecadiaminobutyric acid,    -   or a polycarboxylate in bound form comprising at least 2        carboxylate groups (preferably further comprising a functional        group, which is preferably an additional amino group or a        carboxylic acid group, more preferably an additional carboxylic        acid group),    -   preferably selected from di(glutamic acid), tri(glutamic acid),        tetra(glutamic acid), penta(glutamic acid), hexa(glutamic acid),        hepta(glutamic acid), octa(glutamic acid), nona(glutamic acid),        deca(glutamic acid), undeca(glutamic acid), dodeca(glutamic        acid), trideca(glutamic acid), tetradeca(glutamic acid),        pentadeca(glutamic acid), hexadeca(glutamic acid),        heptadeca(glutamic acid), octadeca(glutamic acid),        nonadeca(glutamic acid), di(aspartic acid), tri(aspartic acid),        tetra(aspartic acid), penta(aspartic acid), hexa(aspartic acid),        hepta(aspartic acid), octa(aspartic acid), nona(aspartic acid),        deca(aspartic acid), undeca(aspartic acid), dodeca(aspartic        acid), trideca(aspartic acid), tetradeca(aspartic acid),        pentadeca(aspartic acid), hexadeca(aspartic acid),        heptadeca(aspartic acid), octadeca(aspartic acid),        nonadeca(aspartic acid), polyethyleneimines, and polyvinylamines

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) is selectedfrom the group comprising, in particular consisting of, in bound form,dilysine, trilysine, tetralysine, pentalysine, hexylysine, heptalysine,octalysine, nonalysine, decalysine, undecalysine, dodecalysine,tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine,heptadecalysine, octadecalysine, nonadecalysine, triornithine,tetraornithine, pentaornithine, hexaornithine, heptaornithine,octaornithine, nonaornithine, decaornithine, undecaornithine,dodecaornithine, tridecaornithine, tetradecaornithine,pentadecaornithine, hexadecaornithine, heptadecaornithine,octadecaornithine, nonadecaornithine, tridiaminobutyric acid,tetradiaminobutyric acid, pentadiaminobutyric acid, hexadiaminobutyricacid, heptadiaminobutyric acid, octadiaminobutyric acid,nonadiaminobutyric acid, decadiaminobutyric acid, undecadiaminobutyricacid, dodecadiaminobutyric acid, tridecadiaminobutyric acid,tetradecadiaminobutyric acid, pentadecadiaminobutyric acid,hexadecadiaminobutyric acid, heptadecadiaminobutyric acid,octadecadiaminobutyric acid, nonadecadiaminobutyric acid, di(glutamicacid), tri(glutamic acid), tetra(glutamic acid), penta(glutamic acid),hexa(glutamic acid), hepta(glutamic acid), octa(glutamic acid),nona(glutamic acid), deca(glutamic acid), undeca(glutamic acid),dodeca(glutamic acid), trideca(glutamic acid), tetradeca(glutamic acid),pentadeca(glutamic acid), hexadeca(glutamic acid), heptadeca(glutamicacid), octadeca(glutamic acid), nonadeca(glutamic acid), di(asparticacid), tri(aspartic acid), tetra(aspartic acid), penta(aspartic acid),hexa(aspartic acid), hepta(aspartic acid), octa(aspartic acid),nona(aspartic acid), deca(aspartic acid), undeca(aspartic acid),dodeca(aspartic acid), trideca(aspartic acid), tetradeca(aspartic acid),pentadeca(aspartic acid), hexadeca(aspartic acid), heptadeca(asparticacid), octadeca(aspartic acid), nonadeca(aspartic acid),polyethyleneimines, and low-molecular weight PEI.

More preferably, a moiety Hyp^(y) of formula (VI) is selected from thegroup comprising, more preferably consisting of, in bound form,trilysine, tetralysine, pentalysine, hexylysine, heptalysine,octalysine, nonalysine, decalysine, undecalysine, dodecalysine,tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine, andheptadecalysine, even more preferably a moiety Hyp^(y) of formula (VI)comprises, preferably consists of, in bound form, trilysine, heptalysineor pentadecalysine.

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) has amolecular weight in the range of from 0.1 kDa to 4 kDa, more preferably0.2 kDa to 2 kDa.

In a further preferred embodiment, each branched moiety Hyp^(y) offormula (VI) has at least 1 branching and is conjugated to at least 2moieties X⁰ of formula (I) and has at most 63 branchings and is at mostconjugated to 64 moieties X⁰ of formula (I), more preferably eachbranched moiety Hyp^(y) of formula (VI) has at least 1 branching and isconjugated to at least 2 moieties X⁰ of formula (I) and has at most 31branchings and is at most conjugated to 32 moieties X⁰ of formula (I).

In a preferred embodiment, Z¹ of formula (VI) comprises a quaternarycarbon, in particular a quaternary carbon of a branching core moiety B,wherein B of formula (VI) is pentarythritol in bound form. Preferably,each A of formula (VI) is independently a PEG-based polymeric chainterminally attached to the quaternary carbon of pentaerythritol via the—CH₂—O— moieties of the branching core moiety pentaerythritol by apermanent covalent linkage, and the distal end of the PEG-basedpolymeric chain is covalently bound to a branched moiety Hyp^(y) offormula (VI), each branched moiety Hyp^(y) of formula (VI) is conjugatedto the moieties X⁰ of formula (I).

In one preferred embodiment, a branched moiety Hyp^(y) of formula (VI)comprises, preferably consists of branched polyamines comprising atleast 2 amine groups. Preferably, the branched polyamine comprising atleast 2 amine groups, comprises one or more lysine residues in boundform. Preferably, each branched moiety Hyp^(y) of formula (VI) has amolecular weight in the range of from 0.1 kDa to 4 kDa, particular 0.2to 2 kDa. In a preferred embodiment, a moiety BA-Hyp^(y))_(n) offormula (VI), wherein n=4, consist of the same or different branchedmoieties Hyp^(y) and that each moiety Hyp^(y) can be chosenindependently. In a preferred embodiment, all moieties Hyp_(y) offormula (VI) are the same.

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) comprises,in particular consists of, between 1 and 32 lysines in bound form,preferably of 1, 3, 7 or 15 lysines in bound form, more preferably of 1,3 or 7 lysines in bound form. Most preferably, Hyp^(y) of formula (VI)comprises, in particular consists of heptalysinyl.

Preferably, the moiety BA-Hyp^(y))_(n) of formula (VI), wherein n ispreferably 4, has a molecular weight in the range of from 1 kDa to 160kDa, more preferably 1 kDa to 80 kDa and even more preferably 10 kDa to40 kDa.

Preferred moieties BA-Hyp^(y))₄ of formula (VI) are selected fromstructures (i-y) to (iii-y):

-   -   wherein    -   dashed lines indicate attachment to a moiety X⁰ of formula (I),        provided that one of m1, m2 is 1 and wherein the carrier is        covalently attached to T in case m1, m2=0,    -   p is an integer of from 5 to 2000, preferably from 10 to 1000,        more preferably from 10 to 500, most preferably from 100 to        1000,    -   q is 1 or 2.

In a preferred embodiment, B of formula (VI) is pentaerythritol.

In another preferred embodiment, Z¹ of formula (I) is a protein carrierwhich comprises, in particular consists of an amino acid sequence of atleast 100 amino acid residues.

In another preferred embodiment, the protein carrier Z¹ of formula (I)is in random coil conformation.

In another preferred embodiment, the protein carrier Z¹ of formula (I)comprises, in particular consists of alanine, serine and prolineresidues.

In the preferred embodiment, the protein carrier Z¹ of formula (I)comprises, in particular consists of an amino acid sequence of at least100 amino acid residues, and

-   -   wherein the amino acid sequence of at least 100 amino acid        residues is in random coil conformation, and,    -   wherein the amino acid sequence of at least 100 amino acid        residues comprises alanine, serine and proline residues.

Preferably, the protein carrier a protein carrier Z¹ of formula (I) iscomposed of an amino acid sequence comprising at least about 100 aminoacid residues, at least 100 amino acid residues, consisting of alanine,serine and proline residues which have a random coil conformation atphysiological conditions. It is understood that the protein carrier Z¹of formula (I) may transiently or temporarily not form a random coil,for example when present in a lyophilisate or dried composition.

In one embodiment the protein carrier Z¹ of formula (I) has a randomcoil conformation with an amino acid sequence of maximally about 3000amino acid residues, preferably of maximally about 1500 amino acidresidues, more preferably of maximally about 900 amino acid residues,even more preferably of maximally about 700 amino acid residues,particularly preferably of maximally about 600 amino acid residues.Thus, the amino acid sequence forming random coil conformation ismaximally about 500 amino acid residues or of maximally about 450 aminoacid residues in length.

Accordingly, the protein carrier Z¹ of formula (I), in particular theamino acid sequence forming random coil conformation of the proteincarrier Z¹ of formula (I) is about 100 to about 3000 amino acid residuesin length.

In particular embodiments said amino acid sequence forming random coilconformation of about 100 to 1000 amino acid residues is ascharacterized herein, i.e. comprising alanine, serine and proline asmain or unique residues as defined below.

The protein carrier moiety Z¹ of formula (I) consists mainly of thethree amino acid residues alanine, serine and proline, and wherein allthree amino acids are present in a protein carrier moiety Z¹ of formula(I), whereby proline residues represent preferably about 4% to about 40%of the protein carrier Z¹ of formula (I). The alanine and serineresidues preferably comprise the remaining at least 60% to 96% of theprotein carrier Z¹ of formula (I). However, as will be detailed hereinbelow said protein carrier Z¹ of formula (I) may also comprise furtheramino acids differing from alanine, serine, and proline, i.e. as minorconstituents.

The term “minor constituent” as used herein means that maximally 10%(i.e. maximally 10 of 100 amino acids) may be different from alanine,serine and proline, preferably maximally 8% (i.e. maximally 8 of 100amino acids) may be different than alanine, serine and proline, morepreferably maximally 6% (i.e. maximally 6 of 100 amino acids) may bedifferent from alanine, serine and proline, even more preferablymaximally 5% (i.e. maximally 5 of 100 amino acids) may be different fromalanine, serine and proline, particularly preferably maximally 4% (i.e.maximally 4 of 100 amino acids) may be different from alanine, serineand proline, more particularly preferably maximally 3% (i.e. maximally 3of 100 amino acids) may be different from alanine, serine and proline,even more particularly preferably maximally 2% (i.e. maximally 2 of 100amino acids) may be different from alanine, serine and proline and mostpreferably maximally 1% (i.e. maximally 1 of 100 of the amino acids)that encode the protein carrier Z¹ of formula (I) may be different fromalanine, serine and proline. Said amino acids different from alanine,serine and proline may be selected from the group of natural orproteinogenic amino-acids consisting of Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Minorconstituents may also be selected from non-naturally occurring aminoacids, such as, for example, hydroxyproline or selenomethionine or othermodified natural amino acids.

The term “at least about 100/150/200/250/300/300/350 (etc) amino acidresidues” is not limited to the concise number of amino acid residuesbut also comprises amino acid stretches that comprise an additional 10%to 20% or comprise 10% to 20% less residues. For example “at least about100 amino acid residues” may also comprise 80 to 100 and about 100 to120 amino acid residues.

In one embodiment, the protein carrier Z¹ of formula (I) comprises aplurality of polymer cassettes wherein said polymer cassettes consist ofAla, Ser, and/or Pro, and wherein no more than 6 consecutive amino acidresidues of the polymer cassettes, preferably of the protein carrier Z¹of formula (I) are identical and wherein said proline residuesconstitute more than 4% and less than 40% of the amino acids of saidprotein carrier Z¹ of formula (I).

In one embodiment, the protein carrier moiety Z¹ of formula (I)comprises, preferably consists of a plurality of amino acid repeats,

-   -   wherein said repeats consist of Ala, Ser, and Pro residues,    -   and wherein no more than 6 consecutive amino acid residues of        the carrier moiety Z¹ of formula (I) are identical.

In a preferred embodiment, said proline residues constitute more than 4%and less than 40% of the amino acids of the protein carrier moiety Z¹ offormula (I).

In a further preferred embodiment, the protein carrier moiety Z¹ offormula (I) comprises, in particular consists of an amino acid sequenceof about 100 to 3000 amino acid residues forming random coilconformation.

The protein carrier Z¹ of formula (I) may comprise a plurality ofidentical polymer cassettes or a plurality of non-identical polymercassettes. Non-limiting examples of polymer cassettes consisting of Ala,Ser and/or Pro residues are provided herein below; see SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 orpeptide fragments or multimers of these sequences. A polymer cassettemay consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more aminoacid residues, wherein each polymer cassette comprises (an) Ala, Ser,and/or Pro residue(s), preferably (an) Ala, Ser, and Pro residue(s).

In one embodiment, the polymer cassette does not comprise more than 100amino acid residues. Preferably, a polymer cassette as defined hereincomprises more than about 4%, preferably more than about 5%, even morepreferably more than about 6%, particularly preferably more than about8%, more particularly preferably more than about 10%, even moreparticularly preferably more than about 15% and most preferably morethan about 20% proline residues. Such polymer cassette as defined hereinpreferably comprises less than about 40% or less than about 35% prolineresidues.

In one embodiment the protein carrier Z¹ of formula (I) is of formula(b):

Ser_(x)[Ala_(y)Ser_(z)]_(v)  (b),

-   -   which formula further comprises proline residues as defined        herein and wherein    -   x is independently selected from integer 0 to 6,    -   each y is independently selected from integer ranging of from 1        to 6,    -   each z is independently selected from integer ranging of from 1        to 6.    -   v is any integer so that the protein carrier Z¹ consists of at        least about 100 amino acid residues, and in particular of at        least about 100 to about 3000 amino acid residues, preferably to        about 2000 and more preferably to about 1000 amino acid        residues.

In one embodiment, all y of formula (b) and z of formula (b) of the vAla_(y) Ser_(z) monomer moieties of formula (b) are identical. Inanother embodiment, the y of formula (b) and z of formula (b) of the vAla_(y) Ser_(z) monomer moieties of formula (b) are different.

In preferred embodiments, the protein carrier Z¹ of formula (I)comprises no more than 5 identical consecutive amino acid residues, morepreferably no more than 4 identical consecutive amino acid residues andmost preferably no more than 3 identical consecutive amino acidresidues.

As already indicated herein above, the protein carrier Z¹ of formula (I)comprises proline residues, wherein said proline residues constitutemore than about 4%, preferably more than about 5%, even more preferablymore than about 6%, particularly preferably more than about 8%, moreparticularly preferably more than about 10%, even more particularlypreferably more than about 15% and most preferably more than about 20%of the amino acids constituting the protein carrier Z¹ of formula (I).Such proline residues may be introduced at any position in formula (b).Preferably, the proline residues may be present in one or more of the vAla_(y) Ser_(z) monomers of formula (b), and they may be present at thesame or at different positions.

In another preferred embodiment, the protein carrier Z¹ of formula (I)comprises more than about 4% but less than about 50%, preferably morethan about 10% but less than about 50% and most preferably more thanabout 20% but less than about 50% alanine residues of the amino acidsconstituting the protein carrier Z¹ of formula (I).

In a further preferred embodiment, the protein carrier Z¹ of formula (I)comprises more than about 4% and less than about 50%, preferably morethan about 10% but less than about 50% and most preferably more thanabout 20% but less than about 50% serine residues of the amino acidsconstituting the protein carrier Z¹ of formula (I).

Accordingly, the protein carrier Z¹ of formula (I) comprises about 35%proline residues, about 50% alanine residues and about 15% serineresidues of the amino acids constituting the protein carrier Z¹ offormula (I). Alternatively, the protein carrier Z¹ of formula (I) maycomprise about 35% proline residues, about 15% alanine residues andabout 50% serine residues of the amino acids constituting the proteincarrier Z¹ of formula (I).

Preferably, the protein carrier Z¹ of formula (I) is comprises one ormore of the following alanine-serine polymer cassettes:

SEQ ID NO: 1 AAAASSASSASSSSSAAASA SEQ ID NO: 2 AASAAASSAAASAAAASASSSEQ ID NO: 3 ASASASASASASSAASAASA SEQ ID NO: 4 SAASSSASSSSAASSASAAASEQ ID NO: 5 SSSSAASAASAAAAASSSAS SEQ ID NO: 6 SSASSSAASSSASSSSASAASEQ ID NO: 7 SASASASASASAASSASSAS SEQ ID NO: 8 ASSAAASAAAASSAASASSS

provided that the protein carrier Z¹ of formula (I) further comprisesproline residues as described herein.

The multimers of these alanine-serine polymer cassettes may form randomcoil conformation in case the resulting amino acid sequence furthercomprises proline residues as defined herein above.

In a preferred embodiment, the protein carrier Z¹ of formula (I)comprises, preferably consists of one or more of the following polymercassettes:

SEQ ID NO: 9 ASPAAPAPASPAAPAPSAPA SEQ ID NO: 10 AAPASPAPAAPSAPAPAAPSSEQ ID No: 11 APSSPSPSAPSSPSPASPSS SEQ ID NO: 15 SAPSSPSPSAPSSPSPASPS

SEQ ID NO:15 corresponds to the herein provided SEQ ID No:11 in acircularly permuted form, wherein the last serine was removed andanother serine was appended as starting amino acid. As a consequence,multimers of this modified sequence possess essentially the sameinternal repeating unit as multimers of the non-modified sequence,except for the very first and the very last residue. Accordingly, SEQ IDNO:15 may be considered as an example of a further polymer cassette forthe protein carrier Z¹ of formula (I). It is clear for the personskilled in the art that also other polymer cassettes and (shorter)peptide fragments or circularly permuted versions of the herein providedamino acid polymers may be used as polymer cassettes for the proteincarrier Z¹ of formula (I).

Yet, even further and illustrative amino acid polymers forming randomcoil conformation may comprise amino acid sequences that may be selectedfrom the group consisting of:

SEQ ID NO: 12 SSPSAPSPSSPASPSPSSPA, SEQ ID NO: 13AASPAAPSAPPAAASPAAPSAPPA, and SEQ ID NO: 14 ASAAAPAAASAAASAPSAAA.

Therefore, preferred polymer cassettes for Z¹ of formula (I) areselected from the following sequences:

(SEQ ID NO: 9) ASPAAPAPASPAAPAPSAPA, (SEQ ID NO: 10)AAPASPAPAAPSAPAPAAPS, (SEQ ID NO: 11) APSSPSPSAPSSPSPASPSS,(SEQ ID NO: 12) SSPSAPSPSSPASPSPSSPA, (SEQ ID NO: 13)AASPAAPSAPPAAASPAAPSAPPA, and (SEQ ID NO: 14) ASAAAPAAASAAASAPSAAA;

-   -   or circular permuted versions or (a) multimer(s) of these        sequences as a whole or parts of these sequences.

In one embodiment, the protein carrier moiety Z¹ of formula (I)comprises at least one amino acid sequence selected from the groupconsisting of:

(SEQ ID NO: 9) ASPAAPAPASPAAPAPSAPA, (SEQ ID NO: 10)AAPASPAPAAPSAPAPAAPS, (SEQ ID NO: 11) APSSPSPSAPSSPSPASPSS,(SEQ ID NO: 12) SSPSAPSPSSPASPSPSSPA, (SEQ ID NO: 13)AASPAAPSAPPAAASPAAPSAPPA, and (SEQ ID NO: 14) ASAAAPAAASAAASAPSAAA;

and circular permuted versions or (a) multimer(s) of these sequences asa whole or parts of these sequences.

Again, also (a) peptide fragment(s) or (a) multimer(s) or circularlypermuted versions of these sequences and the sequences provided hereinabove may be employed as polymer cassettes for the protein carrier Z¹ offormula (I).

Accordingly, the exemplified polymer cassettes may also provide forindividual peptide fragments which may be newly combined to form furtherpolymer cassettes.

In accordance with the above, the protein carrier Z¹ of formula (I) maycomprise a multimer consisting of either one of the amino acid sequenceswith SEQ ID NO:9, 10, 11, 12, 13 or 14 as disclosed herein above or maycomprise a multimer consisting of more than one of amino acid sequencesSEQ ID NO:9, 10, 11, 12, 13 and 14. Furthermore, it is envisaged thatalso peptide fragments or circularly permuted versions of theseexemplified sequences may be used to build up further polymer cassettesof the protein carrier Z¹ of formula (I).

In another embodiment, the protein carrier Z¹ of formula (I) maycomprise a multimer comprising, preferably consisting of a (circular)permutation of the amino acid sequence selected from the groupconsisting of SEQ ID NOs:9, 10, 11, 12, 13, 14, 15 and (a) multimers(s)of these (circular) permutated sequences.

In yet another embodiment, the protein carrier Z¹ of formula (I) maycomprise, preferably consist of a multimer consisting of a peptidefragment/part of the amino acid sequence selected from the groupconsisting of SEQ ID NO: 9, 10, 12, 13, 14, 15 and (a) multimers(s) ofthese exemplified polymer cassettes.

Peptide fragments of these sequences to be employed for the generationof the protein carrier Z¹ of formula (I) may consist of at least 3,preferably of at least 4, more preferably of at least 5, even morepreferably of at least 6, still more preferably of at least 8,particularly preferably of at least 10, more particularly preferably ofat least 12, even more particularly preferably of at least 14,preferably of at least 6, still more preferably of at least 8,particularly preferably of at least 10, more particularly preferably ofat least 12, even more particularly preferably of at least 14, even moreparticularly preferably of at least 16, and most preferably of at least18 consecutive amino acids of the amino acid sequence selected from thegroup consisting of said SEQ ID NOs: 9, 10, 11, 12, 13 and 14.

For example, individual peptide fragments of the polymer cassettes maybe combined to further individual polymer cassettes as long as theabove-identified rules for the overall distribution and amount ofalanine, serine and proline are respected. Again, these polymercassettes may also comprise further amino acid residues, however only asminimal or minor constituents, i.e. maximally 10%, preferably maximally2% of the individual polymer cassette. Said individual polymer cassettesconsist of at least about 100 amino acid residues. Individual polymercassettes may be combined in order to form longer random coil formingamino acid polymers, whereby a maximal length of the protein carrier Z¹of formula (I) is about 3000 amino acids. A preferred minor constituentof the protein carrier Z¹ is lysine.

The pharmaceutical compositions comprising a prostacyclin compound maycomprise one or more excipients. Excipients may be categorized asbuffering agents, isotonicity modifiers, preservatives, stabilizers,anti-adsorption agents, oxidation protection agents,viscosifiers/viscosity enhancing agents, or other auxiliary agents. Insome cases, these ingredients may have dual or triple functions. Thepharmaceutical compositions comprising a prostacyclin compound accordingto the present invention contain one or more excipients, selected fromthe groups consisting of:

-   (i) Buffering agents: physiologically tolerated buffers to maintain    pH in a desired range, such as sodium phosphate, bicarbonate,    succinate, histidine, citrate and acetate, sulphate, nitrate,    chloride, pyruvate. Antacids such as Mg(OH)₂ or ZnCO₃ may be also    used. Buffering capacity may be adjusted to match the conditions    most sensitive to pH stability-   (ii) Isotonicity modifiers: to minimize pain that can result from    cell damage due to osmotic pressure differences at the injection    depot. Glycerin and sodium chloride are examples. Effective    concentrations can be determined by osmometry using an assumed    osmolality of 285-315 mOsmol/kg for serum-   (iii) Preservatives and/or antimicrobials: multidose parenteral    preparations require the addition of preservatives at a sufficient    concentration to minimize risk of patients becoming infected upon    injection and corresponding regulatory requirements have been    established. Typical preservatives include m-cresol, phenol,    methylparaben, ethylparaben, propylparaben, butylparaben,    chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol,    sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and    benzalkonium chloride-   (iv) Stabilizers: Stabilization is achieved by strengthening of the    protein-stabilizing forces, by destabilization of the denatured    state, or by direct binding of excipients to the protein.    Stabilizers may be amino acids such as alanine, arginine, aspartic    acid, glycine, histidine, lysine, proline, sugars such as glucose,    sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol,    salts such as potassium phosphate, sodium sulphate, chelating agents    such as EDTA, hexaphosphate, ligands such as divalent metal ions    (zinc, calcium, etc.), other salts or organic molecules such as    phenolic derivatives. In addition, oligomers or polymers such as    cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA    may be used-   (v) Anti-adsorption agents: Mainly ionic or non-ionic surfactants or    other proteins or soluble polymers are used to coat or adsorb    competitively to the inner surface of the composition's or    composition's container. Suitable surfactants are e.g., alkyl    sulfates, such as ammonium lauryl sulfate and sodium lauryl sulfate;    alkyl ether sulfates, such as sodium laureth sulfate and sodium    myreth sulfate; sulfonates such as dioctyl sodium sulfosuccinates,    perfluorooctanesulfonates, perfluorobutanesulfonates, alkyl benzene    sulfonates; phosphates, such as alkyl aryl ether phosphates and    alkyl ether phosphates; carboxylates, such as fatty acid salts    (soaps) or sodium stearate, sodium lauroyl sarcosinate,    perfluorononanoate, perfluorooctanoate; octenidine dihydrochloride;    quaternary ammonium cations such as cetyl trimethylammonium bromide,    cetyl trimethylammonium chloride, cetylpyridinium chloride,    polyethoxylated tallow amine, benzalkonium chloride, benzethonium    chloride, 5-bromo-5-nitor-1,3-dioxane, dimethyldioctadecylammonium    chloride, dioctadecyldimethylammonium bromide; zwitterionics, such    as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,    cocamidopropyl hydroxysultaine, amino acids, imino acids,    cocamidopropyl betaine, lecithin; fatty alcohols, such as cetyl    alcohol, stearyl alcohol, cetostearyl alcohol, oleyl alcohol;    polyoxyethylene glycol alkyl ethers, such as octaethylene glycol    monododecyl ether, pentaethylene glycol monododecyl ether;    polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers, such    as decyl glucoside, lauryl glucoside, octyl glucoside;    polyoxyethylene glycol octylphenol ethers such as Triton X-100;    polyoxyethylene glycol alkylphenol ethers such as nonoxynol-9;    glycerol alkyl esters such as glyceryl laurate; polyoxyethylene    glycol sorbitan alkyl esters such as polysorbates; sorbitan alkyl    esters; cocamide MEA and cocamide DEA; dodecyl dimethylamine oxide;    block copolymers of polyethylene glycol and polypropylene glycol,    such as poloxamers (Pluronic F-68), PEG dodecyl ether (Brij 35),    polysorbate 20 and 80; other anti-absorption agents are dextran,    polyethylene glycol, PEG-polyhistidine, BSA and HSA and gelatines.    Chosen concentration and type of excipient depends on the effect to    be avoided but typically a monolayer of surfactant is formed at the    interface just above the CMC value-   (vi) Lyo- and/or cryoprotectants: During freeze- or spray drying,    excipients may counteract the destabilizing effects caused by    hydrogen bond breaking and water removal. For this purpose sugars    and polyols may be used but corresponding positive effects have also    been observed for surfactants, amino acids, non-aqueous solvents,    and other peptides. Trehalose is particularly efficient at reducing    moisture-induced aggregation and also improves thermal stability    potentially caused by exposure of protein hydrophobic groups to    water. Mannitol and sucrose may also be used, either as sole    lyo/cryoprotectant or in combination with each other where higher    ratios of mannitol:sucrose are known to enhance physical stability    of a lyophilized cake. Mannitol may also be combined with trehalose.    Trehalose may also be combined with sorbitol or sorbitol used as the    sole protectant. Starch or starch derivatives may also be used-   (vii) Oxidation protection agents: antioxidants such as ascorbic    acid, ectoine, methionine, glutathione, monothioglycerol, morin,    polyethylenimine (PET), propyl gallate, vitamin E, chelating agents    such aus citric acid, EDTA, hexaphosphate, thioglycolic acid-   (viii) Spreading or diffusing agent: modifies the permeability of    connective tissue through the hydrolysis of components of the    extracellular matrix in the intrastitial space such as but not    limited to hyaluronic acid, a polysaccharide found in the    intercellular space of connective tissue. A spreading agent such as    but not limited to hyaluronidase temporarily decreases the viscosity    of the extracellular matrix and promotes diffusion of injected    drugs.-   (ix) Other auxiliary agents: such as wetting agents, viscosity    modifiers, antibiotics, hyaluronidase. Acids and bases such as    hydrochloric acid and sodium hydroxide are auxiliary agents    necessary for pH adjustment during manufacture.

In a general embodiment the pharmaceutical composition comprising aprostacyclin compound of the present invention in either dry or liquidform may be provided as a single or multiple dose composition.

In one embodiment of the present invention, the liquid or drypharmaceutical composition comprising a prostacyclin compound isprovided as a single dose, meaning that the container in which it issupplied contains one pharmaceutical dose.

Alternatively, the liquid or dry pharmaceutical composition aprostacyclin compound is a multiple dose composition, meaning that thecontainer in which it is supplied contains more than one therapeuticdose, i.e., a multiple dose composition contains at least 2 doses. Suchmultiple dose composition comprising a prostacyclin compound can eitherbe used for different patients in need thereof or can be used for onepatient, wherein the remaining doses are stored after the application ofthe first dose until needed.

In another aspect of the present invention the pharmaceuticalcomposition comprising a prostacyclin compound is comprised in acontainer. Suitable containers for liquid or dry compositions are, forexample, syringes, vials, vials with stopper and seal, ampoules, andcartridges. In particular, the liquid or dry composition comprising aprostacyclin compound according to the present invention is provided ina syringe. If the pharmaceutical composition comprising a prostacyclincompound is a dry pharmaceutical composition the container preferably isa dual-chamber syringe. In such embodiment, said dry pharmaceuticalcomposition is provided in a first chamber of the dual-chamber syringeand reconstitution solution is provided in the second chamber of thedual-chamber syringe.

Prior to applying the dry composition comprising a prostacyclin compoundto a patient in need thereof, the dry composition is reconstituted.Reconstitution can take place in the container in which the drycomposition comprising a prostacyclin compound is provided, such as in avial, syringe, dual-chamber syringe, ampoule, and cartridge.Reconstitution is done by adding a predefined amount of reconstitutionsolution to the dry composition. Reconstitution solutions are sterileliquids, such as water or buffer, which may contain further additives,such as preservatives and/or antimicrobials, such as, for example,benzyl alcohol and cresol. Preferably, the reconstitution solution issterile water. When a dry composition is reconstituted, it is referredto as a “reconstituted pharmaceutical composition” or “reconstitutedcomposition”.

An additional aspect of the present invention relates to the method ofadministration of a reconstituted or liquid pharmaceutical compositioncomprising a prostacyclin compound of the present invention. Thepharmaceutical composition comprising a prostacyclin compound may beadministered by methods of inhalation, injection or infusion, includingintradermal, subcutaneous, intramuscular, intravenous, intraosseous, andintraperitoneal. Preferably, the pharmaceutical composition comprising aprostacyclin compound is administered subcutaneously.

The preferred method of administration for dry pharmaceuticalcompositions comprising a prostacyclin compound of the present inventionis via inhalation.

In another embodiment, a reconstituted or liquid pharmaceuticalcomposition comprising a prostacyclin compound of the present inventionis administered via a first method of administration and a secondreconstituted or liquid pharmaceutical composition comprising aprostacyclin compound of the present invention is administered via asecond method of administration, either simultaneously or consecutively.Said first and second method of administration can be any combination oftopical, enteral administration, parenteral administration, inhalation,injection, or infusion, intraarticular, intradermal, subcutaneous,intramuscular, intravenous, intraosseous, and intraperitoneal,intrathecal, intracapsular, intraorbital, intracardiac, transtracheal,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,intraventricular or intrasternal administration.

A further aspect is a method of preparing a reconstituted compositioncomprising a therapeutically effective amount of a prostacyclin compoundof the present invention, and optionally one or more pharmaceuticallyacceptable excipients, the method comprising the step of

-   -   contacting the pharmaceutical composition comprising a        prostacyclin compound of the present invention with a        reconstitution solution.

Another aspect is a reconstituted pharmaceutical composition comprisinga therapeutically effective amount of a prostacyclin compound of thepresent invention, and optionally one or more pharmaceuticallyacceptable excipients.

Another aspect of the present invention is the method of manufacturing adry composition of a prostacyclin compound. In one embodiment, such drycomposition is made by

-   (i) admixing the prostacyclin compound with one or more excipients,-   (ii) transferring amounts equivalent to single or multiple doses    into a suitable container,-   (iii) drying the composition in said container, and-   (iv) sealing the container.

Suitable containers are vials, syringes, dual-chamber syringes,ampoules, and cartridges.

Another aspect of the present invention is a kit of parts.

If the administration device is simply a hypodermic syringe then the kitmay comprise the syringe, a needle and a container comprising the drypharmaceutical composition comprising a prostacyclin compound for usewith the syringe and a second container comprising the reconstitutionsolution.

If the pharmaceutical composition is a liquid composition then the kitmay comprise the syringe, a needle and a container comprising the liquidcomposition comprising a prostacyclin compound for use with the syringe.

In more preferred embodiments, the injection device is other than asimple hypodermic syringe and so the separate container withreconstituted or liquid prostacyclin compound is adapted to engage withthe injection device such that in use the liquid composition in thecontainer is in fluid connection with the outlet of the injectiondevice. Examples of administration devices include but are not limitedto hypodermic syringes and pen injector devices. Particularly preferredinjection devices are the pen injectors in which case the container is acartridge, preferably a disposable cartridge. Optionally, the kit ofparts comprises a safety device for the needle which can be used to capor cover the needle after use to prevent injury.

A preferred kit of parts comprises a needle and a container containingthe composition according to the present invention and optionallyfurther containing a reconstitution solution, the container beingadapted for use with the needle. Preferably, the container is adual-chamber syringe.

In another aspect, the invention provides a cartridge comprising apharmaceutical composition comprising a prostacyclin compound ashereinbefore described for use with a pen injector device. The cartridgemay contain a single dose or multiplicity of doses of the prostacyclincompound.

Yet another aspect of the present invention is a pharmaceuticalcomposition comprising a prostacyclin compound of the present inventionor a pharmaceutical composition of the present invention for use as amedicament.

In a further embodiment, the present invention relates to a prostacyclincompound or pharmaceutical composition of the present invention for thepreparation of a medicament, in particular for the preparation of amedicament with one or more of the features described herein.

In case a prostacyclin compound comprised in the pharmaceuticalcompositions according to the invention contain one or more acidic orbasic groups, the invention also comprises their correspondingpharmaceutically or toxicologically acceptable salts, in particulartheir pharmaceutically utilizable salts. Thus, the pharmaceuticalcomposition comprising a prostacyclin compound according to theinvention which contains acidic groups can be used according to theinvention, for example, as alkali metal salts, alkaline earth metalsalts or as ammonium salts. More precise examples of such salts includesodium salts, potassium salts, calcium salts, magnesium salts or saltswith ammonia or organic amines such as, for example, ethylamine,ethanolamine, triethanolamine or amino acids. Pharmaceuticalcompositions comprising a prostacyclin compound according to theinvention which contain one or more basic groups, i.e. groups which canbe protonated, can be present and can be used according to the inventionin the form of their addition salts with inorganic or organic acids.Examples for suitable acids include hydrogen chloride, hydrogen bromide,phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, aceticacid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formicacid, propionic acid, pivalic acid, diethylacetic acid, malonic acid,succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid,sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid,isonicotinic acid, citric acid, adipic acid, and other acids known tothe person skilled in the art. If the pharmaceutical compositionscomprising a prostacyclin compound according to the inventionsimultaneously contain acidic and basic groups in the molecule, theinvention also includes, in addition to the salt forms mentioned, innersalts or betaines (zwitterions). The respective salts can be obtained bycustomary methods which are known to the person skilled in the art like,for example by contacting these with an organic or inorganic acid orbase in a solvent or dispersant, or by anion exchange or cation exchangewith other salts. The present invention also includes all salts of theprodrugs which, owing to low physiological compatibility, are notdirectly suitable for use in pharmaceuticals but which can be used, forexample, as intermediates for chemical reactions or for the preparationof pharmaceutically acceptable salts.

Yet another aspect of the present invention is a method of treating,controlling, delaying or preventing in a mammalian patient, preferablyin a human, in need of the treatment of one or more conditionscomprising administering to said patient a therapeutically effectiveamount of a prostacyclin compound or a pharmaceutical compositioncomprising a prostacyclin compound of the present invention or apharmaceutically acceptable salt thereof.

The invention further relates to a method for treating pulmonaryhypertension, wherein the method comprises the step of subcutaneous orintramuscular administration, to a patient with pulmonary hypertension,of a pharmaceutical composition comprising

-   -   (a) a polymer carrier-linked prostacyclin prodrug, and        optionally one or more pharmaceutically acceptable excipients,        or    -   (b) a prostacyclin compound and at least one polymer, and        optionally one or more pharmaceutically acceptable excipients,        wherein the pharmaceutical composition releases therapeutically        effective amounts of free prostacyclin compound for a period of        time of at least 12 hours.

In a preferred embodiment, the pharmaceutical composition formulation of(b) comprises from about 0.05 to about 10 weight percent of theprostacyclin compound, more preferably 0.01 to 5 weight percent of theprostacyclin compound and most preferably from 0.1 to about 2 weightpercent of the prostacyclin compound and from about 0.5 to about 20weight percent total polymer content, preferably from about 1 to about10 weight percent total polymer content and most preferably from about 1to about 7 weight percent total polymer.

In a preferred embodiment, the pharmaceutical compositions of thepresent invention are sustained release formulations of a prostacyclin.

The prostacyclin compounds, and pharmaceutical compositions comprising aprostacyclin compound, and pharmaceutical compositions comprising aprostacyclin compound for use of the present invention can be used forthe treatment and/or prevention of, for example, pulmonary hypertension,ischemic diseases (e.g. peripheral vascular disease including peripheralarterial disease, Raynaud's phenomenon including Raynaud's disease andRaynaud's syndrome, scleroderma including systemic sclerosis, myocardialischemia, ischemic stroke, renal insufficiency), ischemic ulcersincluding digital ulcers, heart failure (including congestive heartfailure), portopulmonary hypertension, interstitial lung disease,idiopathic pulmonary fibrosis, conditions requiring anticoagulation(e.g., post MI, post cardiac surgery), thrombotic microangiopathy,extracorporeal circulation, central retinal vein occlusion,atherosclerosis, inflammatory diseases (e.g., COPD, psoriasis),hypertension (e.g., preeclampsia), reproduction and parturition, canceror other conditions of unregulated cell growth, cell/tissue preservationand other emerging therapeutic areas where prostacyclin treatmentappears to have a beneficial role.

Therefore, the present invention relates to a pharmaceutical compositionof the present invention for use in the treatment or prevention ofpulmonary hypertension, ischemic diseases, preferably peripheralvascular disease including peripheral arterial disease, Raynaud'sphenomenon including Raynaud's disease and Raynaud's syndrome,scleroderma including systemic sclerosis, myocardial ischemia, ischemicstroke, renal insufficiency, ischemic ulcers including digital ulcers,heart failure, in particular congestive heart failure, portopulmonaryhypertension, interstitial lung disease, idiopathic pulmonary fibrosis,conditions requiring anticoagulation, in particular post MI, postcardiac surgery, thrombotic microangiopathy, extracorporeal circulation,central retinal vein occlusion, atherosclerosis, inflammatory diseases,in particular COPD, psoriasis, hypertension, in particular preeclampsia,reproduction and parturition, cancer or other conditions of unregulatedcell growth, cell/tissue preservation and other emerging therapeuticareas where prostacyclin treatment appears to have a beneficial role.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated hereinafter by drawings and the workingexamples, the drawings and working examples serving merely forillustration but not restricting the invention.

FIG. 1 shows the treprostinil release in buffer and buffered rat plasmaat different time points expressed as % treprostinil release compared tototal treprostinil content (see Example 29).

FIG. 2 shows a prolonged duration of circulation of treprostinilconjugate 25 for more than two weeks in monkeys after subcutaneousinjection (see Example 30).

FIG. 3 shows a single dose iv injection of compound 25 and subsequentplasma analysis for total treprostinil and carrier content (see Example30).

FIG. 4 shows a single dose iv injection of compound 25 and subsequentplasma analysis for free treprostinil (see Example 32).

FIG. 5 shows a single dose subcutaneous injection of compound 25 andsubsequent plasma analysis for free treprostinil (see Example 32).

OPERATIVE EXAMPLES

The subject matter of the present invention is elucidated in more detailbelow, using examples, without any intention that the subject matter ofthe invention should be confined to these exemplary embodiments.

Materials, Methods and Analytics: Product Purification

Normal phase purification was performed on a Biotage “Isolera one”purification system Biotage AB, Sweden. Biotage KP-Sil silicacartridges. Gradients of Heptane/Ethylacetate orDichloromethane/Methanol were used. Products were detected and collectedat 254 and 280 nm.

For preparative RP-HPLC, a Waters 600 controller and a 2487 DualAbsorbance Detector was used equipped with a Waters XBridge™ BEH300 PrepC18 5 μm, 150×10 mm, flow rate 6 ml/min, or Waters XBridge™ BEH300 PrepC18 10 μm, 150×30 mm, flow rate 40 ml/min. Gradients of eluents A (watercontaining 0.05% TFA v/v or 0.01% HCl v/v) and B (acetonitrilecontaining 0.05% TFA v/v or 0.01% HCl v/v) were used.

HPLC fractions containing product were pooled and lyophilized if notstated otherwise.

Automated Flash Chromatography

Automated Flash Chromatography was performed on a Biotage “Isolera one”purification system Biotage AB, Sweden, using Biotage KP-Sil silicacartridges. Products were detected and collected at 254 and 280 nm.

LC/MS Analytics

Analytical RP-HPLC/ESI-MS was performed on waters equipment consistingof a 2695 sample manager, a 2487 Dual Absorbance Detector, and a ZQ 4000ESI instrument equipped with a 5 μm Reprosil Pur 300 Å ODS-3 column(75×1.5 mm) (Dr. Maisch, Ammerbuch, Germany; flow rate: 350 μl/min,typical gradient: 10-90% MeCN in water, 0.05% TFA over 5 min) or on aWaters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQOrbitrap Discovery high resolution/high accuracy mass spectrometerequipped with a Waters ACQUITY UPLC BEH300 C18 RP column (2.1×50 mm, 300Å, 1.7 μm, flow: 0.25 mL/min; solvent A: UP-H₂0+0.04% TFA, solvent B:UP-Acetonitrile+0.05% TFA.

RP-UPLC/ESI-MS was performed on Waters/Thermo equipment consisting of aWaters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQOrbitrap Discovery high resolution/high accuracy mass spectrometerequipped with a ACQUITY UPLC® BEH300 C18 RP column (Waters Corporation,2.1×50 mm, 300 Å, 1.7 μm, Flow: 0.25 mL/min; solvent A: UP-H₂0+0.04%TFA, solvent B: UP-MeCN+0.05% TFA.

Typical gradients for determination of released treprostinil fromTransCon 5 kDa PEG linker treprostinil are: 0.25 mL flow rate, gradient:30-50% B over 10 min

RP-HPLC Purification:

For preparative RP-HPLC a Waters 600 controller and a 2487 DualAbsorbance Detector was used equipped with the following columns: WatersXBridge™ BEH300 Prep C18 5 μm, 150×10 mm, flow rate 6 ml/min, or WatersXBridge™ BEH300 Prep C18 10 μm, 150×30 mm, flow rate 40 ml/min. Lineargradients of solvent system A (water containing 0.05% TFA v/v or 0.01%HCl v/v) and solvent system B (acetonitrile containing 0.05% TFA v/v or0.01%

HCl v/v)

Typical gradients for purification procedures are:

-   -   6 mL/min flow rate, solvent A: H₂0+0.05% TFA, solvent B:        MeCN+0.05% TFA, typical gradient: 1-95% B over 14 min    -   6 mL/min flow rate, solvent A: H₂0+0.05% TFA, solvent B:        MeCN+0.05% TFA, typical gradient: 10-80% B over 14 min    -   40 mL/min flow rate, solvent A: H₂0+0.05% TFA, solvent B:        MeCN+0.05% TFA, typical gradient: 40-95% B over 14 min

HPLC fractions containing product were pooled and lyophilized if notstated otherwise.

Chemicals and Drug Substances:

Treprostinil acid was purchased from Shanghai Techwell BiopharmaceuticalCo., Ltd., Shanghai, Peoples Republic of China or ChirogateInternational Inc. Yangmei, Taiwan. 6-(S-Tritylmercapto)hexanoic acidwas purchased from Polypeptide, Strasbourg, France.Cis-cyclohexanedicarboxylic anhydride was purchased from Alfa Aesar GmbH& Co KG, Karlsruhe, Germany. 2-Chlorotrityl chloride resin (1%,Novabiochem® DVB) was obtained from Merck Biosciences GmbH, Germany.6-(S-Tritylsulfanyl)-hexaneamine was synthesized according to WO-A2009/133137. PEGs used in this work were acquired from NOF Europe N.V.,Grobbendonk, Belgium. All other chemicals were purchased from SigmaAldrich GmbH, Taufkirchen, Germany. Water and acetonitrile foranalytical RP-HPLC were purchased from Biosolve B.V. and TFA from Thermoscientific.

Example 1 Benzyl Protection of 3-Hydroxybutanoic Acid 1

3-Hydroxybutanoic acid 1 (434 mg, 4.17 mmol) was dissolved in THF (10mL) and BnBr (700 μL, 5.89 mmol) and Cs₂CO₃ (2.5 g, 7.67 mmol) wereadded. The reaction mixture was refluxed in a sealed tube for 4-6 hours.After cooling down to room temperature the reaction mixture wasfiltrated and the residue was washed several times with EtOAc. Theorganic solvents were removed and the product was purified by automatedflash chromatography on silica in one portion (SNAP 25 g cartridge, flow30 ml/min, solvent A: DCM, solvent B: MeOH; gradient: 0-5% B over 19 CV)to remove starting material and obtain desired benzyl protected3-hydroxybutanoic acid 2 as yellow oil.

Yield: 361 mg (45%)

MS: m/z 217.1=[M+Na]⁺ (MW+Na calculated=217.2).

Example 2 Coupling Reaction of Benzylated 3-Hydroxybutanoic Acid 2 withTreprostinil

Treprostinil acid (10.5 mg, 0.0268 mmol) was dissolved in DCM (4.5 mL)and DCC (9.4 mg, 0.0455 mmol), HOBT (7.5 mg, 0.0489 mmol) and DMAP (7.5mg, 0.0613 mmol) were added to the solution. Then benzylated3-hydroxybutanoic acid 2 (15 mg, 0.0772 mmol) was dissolved in DCM (0.5mL) and added to the reaction mixture. The mixture was stirred at RTuntil the consumption was complete (analytical RP-HPLC). Volatilesolvents were removed in vacuo and the residue was purified over a smallsilica column (3 ml silica, DCM/MeOH (100:0)-DCM/MeOH (95:5) to obtainthe desired linker treprostinil 3 as yellow oil.

Yield: 8 mg (50%)

MS: m/z 589.3=[M+Na]⁺ (MW+Na calculated=589.7)

Example 3 Hydrogenation Reaction of Benzylester 3

Benzylester 3 (13 mg, 0.0229 mmol) was dissolved in EtOAc (4 Å MS, 2 mL)and 5% palladium on charcoal (5% Pd, 15 mg) was added. Hydrogen wasbubbled through the solution for 30 min. The reaction mixture wasstirred further 12.5 h under hydrogen atmosphere until the consumptionwas complete (analytical RP-HPLC). The mixture was filtered over celiteand washed several times with EtOAc. Organic solvents were removed invacuo and the residue was purified using RP-HPLC (solvent A: H₂O with0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 1-95% B over 20min, flow: 6 mL/min). The product containing fractions were pooled andlyophilized to obtain 4 as white solid.

Yield: 1.9 mg (29%).

MS: m/z 499.3=[M+Na]⁺ (MW+Na calculated=499.6).

Example 4 Coupling Reaction of Linear PEG 5 kDa Amine with LinkerTreprostinil 4

Linker treprostinil 4 (1.9 mg, 3.98 μmol) and linear PEG 5 kDa amine (86mg, 17.2 μmol) were dissolved in THF/MeCN (4 Å MS; 1.5 mL:0.5 mL) andEt₃N (40 μL), a catalytic amount of DMAP and T3P (50% in EtOAc, 50 μL,73.2 μmol) were successively added. The reaction mixture was allowed tostir at rt for 12 h. The reaction mixture was diluted with 20 μL H₂O andvolatile solvents were removed in vacuo. The residue was purified usingRP-HPLC (solvent A: H₂O with 0.05% TFA, solvent B: MeCN with 0.05% TFA,gradient: 10-80% B over 20 min, flow: 6 mL/min). The product containingfractions were pooled and lyophilized to obtain TransCon PEG linkertreprostinil 5 as white solid.

Yield: 12.5 mg (58%).

MS: m/z 1378.6=[M+4H]⁴⁺ (calculated=1378.9) for one representative peakin the polymer distribution.

Example 5 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil 5

TransCon PEG linker treprostinil 5 (0.5-1.5 mg) was incubated in pH 7.4hydrolysis buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05% Tween-20, 1mL) at 37° C. and aliquots were analyzed by UPLC at various time pointsfor released treprostinil.

Half Life Determination of Hydrolysis Kinetics of TransCon PEG LinkerTreprostinil 5:

The percentage of released treprostinil after incubation at pH 7.4 and37° C. for a given time period was determined by integrating thecorresponding peaks (released material versus conjugate) in the RP-UPLCchromatogram. The data as shown in table 1 were subsequently plottedagainst time. By using a first order kinetics fit a half life of 4.20 dfor the treprostinil release from 5 was obtained.

TABLE 1 entry Incubation time [d] released treprostinil [%] 1 0.000 2 20.83 5 3 1.11 18 4 1.81 27 5 2.06 29 6 5.13 59 7 6.10 64 8 8.80 77 911.90 86

Example 6 Synthesis of Intermediates 6a/6b

6-(S-Tritylsulfanyl)-hexaneamine (for synthesis see WO-A 2009/133137)(507 mg, 1.35 mmol) was dissolved in DCM (4 ml) andcis-1,2-cyclohexanedicarboxylic anhydride (251 mg, 1.63 mmol) was addedto the reaction mixture at RT. DIPEA (0.70 mL, 4.06 mmol) was added andthe mixture was stirred at RT until complete consumption of6-(S-Tritylsulfanyl)-hexaneamine (LC/MS). Volatile solvents were removedin vacuo, the residue was dissolved in H₂O/MeCN (6:1, 18 mL) and theproduct was purified by RP-HPLC (solvent A: H₂O with 0.05% TFA, solventB: MeCN with 0.05% TFA, gradient: 40-95% B over 16 min, flow: 40ml/min). The pooled fractions were neutralized with sat. NaHCO₃ soln.(pH approx. 6) and the organic solvents were removed in vacuo. Theremaining aqueous phase was extracted twice with DCM. Combined organiclayers were dried with MgSO₄ and the solvent was removed in vacuoobtaining 6a/6b as a racemic mixture.

Yield: 580 mg (81%).

MS: m/z 552.23=[M+Na]⁺ (MW+Na calculated=552.62 g/mol).

Example 7 Synthesis of Intermediates 7a/7b

N-Boc-1,6-hexanediamine (270 mg, 1.25 mmol) was dissolved in DMF (2 ml)and cis-1,2-cyclohexanedicarboxylic anhydride (231 mg, 1.50 mmol) wasadded to the reaction mixture at RT. DIPEA (0.65 mL, 3.76 mmol) wasadded and the mixture was stirred at RT until consumption ofN-Boc-1,6-hexanediamine (LC/MS). The reaction mixture was diluted withH₂O/MeCN (9:1) and the product was purified by RP-HPLC (solvent A: H₂Owith 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 10-80% B over16 min, flow: 40 ml/min). The pooled fractions were neutralized withsat. NaHCO₃ soln. (pH approx. 6) and the organic solvents were removedin vacuo. The remaining aqueous phase was extracted several times withDCM. The organic layers were dried with MgSO₄ and the solvent wasremoved in vacuo obtaining 7a/7b as a racemic mixture.

Yield: 410 mg (88%).

MS: m/z 371.39=[M+H]⁺ (MW+H calculated=371.27 g/mol).

Example 8 Synthesis of Dmob Protected Treprostinil 8

Treprostinil (61 mg, 0.156 mmol) was dissolved in toluene (dry,molecular sieve, 2.5 ml) and silylation reagent BSA (0.6 mL, 0.245 mmol)was added. The reaction mixture was stirred for 12 h at RT. Volatilesolvents were removed in vacuo and the TMS protected treprostinil wasused without further purification.

TMS protected treprostinil was dissolved in DCM (2.5 mL) and H₂O (60μL). DMAP (76 mg, 0.624 mmol), EDC.HCl (119 mg, 0.624 mmol) andDmob-alcohol (105 mg, 0.624 mmol) dissolved in DCM (1 ml) were added.The reaction mixture was stirred at RT until reaction was complete(LC/MS). The solution was diluted with DCM and quenched by addition of0.1 N HCl solution saturated with NaCl. The aqueous phase was extractedseveral times with DCM. Combined organic layers were dried with MgSO₄and the solvent was removed in vacuo obtaining crude product 8. Crudeproduct was purified using RP-HPLC (solvent A: H₂O with 0.05% TFA,solvent B: MeCN with 0.05% TFA, gradient: 35-85% B over 16 min, flow: 40ml/min). Combined HPLC fractions were adjusted to a pH of approx. 7 byadding sat. NaHCO₃ soln. MeCN was removed in vacuo. The remaining H₂Olayer was extracted several times with DCM and the combined organicphases were dried with MgSO₄, filtered and the solvent was removed invacuo obtaining product 8 as colorless solid.

Yield: 69 mg (82%).

MS: m/z 563.20 g/mol=[M+Na]⁺ (MW+Na calculated=563.67 g/mol).

Example 9 Synthesis of Treprostinil Linker Thiol

Carboxylic acid 6a/6b (147 mg, 0.277 mmol), EDC.HCl (53 mg, 0.277 mmol)and DMAP (34 mg, 0.277 mmol) were dissolved in 0.5 mL DCM. Dmobprotected treprostinil 8 (43 mg, 0.08 mmol) was dissolved in 0.5 mL DCMand added to the reaction mixture. The mixture was stirred at RT untilconsumption of 8 was complete (over night, LC/MS). Volatile solventswere removed in vacuo. The residue was dissolved in HFIP (2 mL), TFA(100 μL) and TES (50 μL) and stirred for 30 min at RT (LC/MS). Volatileswere removed in vacuo. The residue was dissolved in H₂O/MeCN (9/1, 0.05%TFA, 2 mL) and the mixture of four possible isomers was purified byRP-HPLC (solvent A: H₂O with 0.05% TFA, solvent B: MeCN with 0.05% TFA,gradient: 60-85% B over 16 min, flow: 6 mL/min). Product isomers elutedas three separable peaks. Fractions containing the peak with theshortest elution time (compound “9x”) were pooled and used in thePEGylation step without further processing. Structural assignment of 9xto the possible isomers 9a, 9b, 9c or 9d was not performed in thisexperiment. Yield of 9x was determined by using Ellman test.

Yield: 8.1 mg (26%)

MS: m/z 682.21 g/mol=[M+Na]⁺ (MW+Na calculated=682.40 g/mol).

Example 10 Synthesis of Treprostinil Linker Amine

Carboxylic acid 7a/7b (50 mg, 0.134 mmol), EDC HCl (26 mg, 0.134 mmol)and DMAP (16 mg, 0.134 mmol) were dissolved in DCM (0.3 mL). Dmobprotected treprostinil 8 (36 mg, 0.066 mmol) was dissolved in DCM (0.5mL) and added to the reaction mixture. The mixture was stirred at RTuntil the consumption was complete (LC/MS). Volatile solvents wereremoved in vacuo. The residue was dissolved in H₂O/MeCN (9/1, 0.05% TFA,2 mL) and the mono coupling products (treprostinil coupled to one 7a/7bmolecule) were separated from the double coupling products (treprostinilcoupled to two 7a/7b molecules) by RP-HPLC: Thermo Fisher Hypersil GoldPFP column, 150×10 mm, solvent A: H₂O with 0.05% TFA, solvent B: MeCNwith 0.05% TFA, gradient: 35-55% B over 16 min, flow: 6 mL/min. HPLCfractions containing mono coupling products were pooled and lyophilized.Lyophilizate was dissolved in HFIP (0.9 mL), DCM (0.1 mL), TFA (100 μL)and TES (20 μL) and stirred for 10 min at RT. Volatiles were removed invacuo, the residue was dissolved in H₂O/MeCN (9/1, 0.05% TFA, 2 mL) andthe and the mixture of four possible isomers was purified by RP-HPLC(solvent A: H₂O with 0.05% TFA, solvent B: MeCN with 0.05% TFA,gradient: 35-55% B over 16 min, flow: 6 mL/min). Product isomers elutedas three separable peaks. Fractions containing the peak with theshortest elution time (compound “10x”) were pooled and used in thePEGylation step without further processing. Structural assignment of the10x to the possible isomers 10a, 10b, 10c or 10d was not performed inthis experiment. Yield of 10x was estimated by HPLC by using atreprostinil calibration curve (280 nm).

Yield: 3.0 mg

MS: m/z 643.28 g/mol=[M+Na]⁺ (MW+Na calculated=643.45 g/mol).

Example 11 PEGylation Reaction of Treprostinil Linker Amine with LinearPEG 5 kDa NHS

To treprostinil linker amine 10x (0.6 mg, 1 μmol in solution, MeCN/H₂O,0.05% TFA, 5 mL) linear PEG 5 kDa NHS (23 mg, 4.6 mmol) was added. Thesolution was neutralized by addition of 0.5 M pH 7.4 buffer (0.5 Mphosphate, 0.6 mL). H₂O (1 mL) was added for obtaining a clear solution,and reaction mixture was incubated at RT for 1 h. Then the reactionmixture was purified by RP-HPLC (solvent A: H₂O with 0.01% HCl, solventB: MeCN with 0.01% HCl, gradient: 10-70% B over 16 min, flow: 6 mL/min)to obtain after lyophilization TransCon linear 5 kDa PEG treprostinil11.

Yield: 3 mg Example 12 PEGylation Reaction of Treprostinil Linker Thiolwith Linear PEG 40 kDa Maleimide

To the treprostinil linker thiol 9x (6.2 mg, 9.42 μmol) solution inMeCN/H₂O (0.05% TFA, 87 mL) linear PEG 40 kDa maleimide (463 mg, 11.3μmol) was added. The solution was neutralized by addition of pH 7.4buffer (0.5 M phosphate, 4.4 mL). After 1 h incubation time anotherportion of linear 40 kDa Mal-PEG (73 mg, 178 μmol) and H₂O (5 mL) wasadded and the reaction solution was incubated for another 1.5 h. Thereaction mixture was purified by RP-HPLC (solvent A: H₂O with 0.01% HCl,solvent B: MeCN with 0.01% HCl, gradient: 30-50% B over 16 min, flow: 40mL/min) to obtain after lyophilization TransCon linear 40 kDa PEGtreprostinil 12.

Yield: 321 mg (82%)

Example 13 PEGylation Reaction of Treprostinil Linker Thiol with 4-ArmPEG 20 kDa Maleimide

To the treprostinil linker thiol 9x (2.54 mg, 3.84 μmol) solution inMeCN/H₂O (0.05% TFA, 5.7 mL) 4-arm PEG 20 kDa maleimide (21 mg, 0.98μmol) was added. The solution was neutralized by addition of pH 7.4buffer (0.5 M phosphate, 3.0 mL). H₂O (3 mL) was added until thereaction mixture became a clear solution again. The reaction mixture wasincubated at RT for 2 h and then purified by RP-HPLC (solvent A: H₂Owith 0.01% HCl, solvent B: MeCN with 0.01% HCl, gradient: 45-85% B over16 min, flow: 40 mL/min) to obtain after lyophilization TransCon 4-armPEG 20 kDa treprostinil 13.

Yield: 14 mg (66%).

Example 14 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compounds 11 and 12

Release kinetics were determined according to Example 5. A treprostinilrelease half life time of 4.3 days (±0.7 days) was obtained forcompounds 11 and 12.

Example 15 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compound 13

TransCon PEG linker treprostinil 13 (2.5 mg) was incubated in pH 7.4hydrolysis buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05% Tween-20, 1mL) at 37° C. and aliquots were analyzed by UPLC at various time pointsfor released treprostinil. The percentage of released treprostinil wasdetermined in relation to the area of treprostinil after totalhydrolysis of an aliquot (50 μl hydrolysis solution and 25 μl 5 N NaOHwere mixed for 20 min. 25 μl AcOH was added and the resulting solutionwas analyzed by LCMS).

By using a first order kinetics fit, a half life of 5 d for treprostinilrelease from 13 was obtained.

Example 16 Synthesis of Building Block 14

Building block 14 was synthesized according to the following scheme:

Mmt-chloride (3 g, 9.71 mmol) was dissolved in DCM (20 mL) and addeddropwise to a solution of ethylenediamine (6.5 mL, 97.1 mmol) in DCM (20mL). After two hours the solution was poured into diethyl ether (300 mL)and washed three times with 30/1 (v/v) brine/0.1 M NaOH solution (50 mleach) and once with brine (50 mL). The organic phase was dried overNa₂SO₄ and volatiles were removed under reduced pressure. Mmt-protectedamine (3.18 g, 9.56 mmol) was used in the next step without furtherpurification.

The Mmt-protected amine (3.18 g, 9.56 mmol) was dissolved in anhydrousDCM (30 mL). 6-(S-Tritylmercapto)hexanoic acid (4.48 g, 11.47 mmol),PyBOP (5.96 g, 11.47 mmol) and DIPEA (5.0 mL, 28.68 mmol) were added andthe mixture was agitated for 30 min at RT. The solution was diluted withdiethyl ether (250 mL) and washed three times with 30/1 (v/v) brine/0.1M NaOH solution (50 mL each) and once with brine (50 mL). The organicphase was dried over Na₂SO₄ and volatiles were removed under reducedpressure. Amide was purified by flash chromatography eluting withheptane/ethyl acetate containing 0.02% (v/v) diethylmethylamine

Yield: 5.69 g (8.07 mmol).

MS: m/z 705.4=[M+H]⁺ (MW=705.0).

Amide (3.19 g, 4.53 mmol) was dissolved in anhydrous THF (50 mL) andBH₃.THF (1 M solution, 8.5 mL, 8.5 mmol) was added. Solution was stirredfor 16 h at RT. Further BH₃.THF (1 M solution, 14 mL, 14 mmol) was addedand stirred for further 16 h at RT. The reaction was quenched byaddition of methanol (8.5 mL). N,N-dimethyl-ethylenediamine (3 mL, 27.2mmol) was added, the solution was heated to reflux and stirred for 3 h.Reaction mixture was allowed to cool down to RT and was then dilutedwith ethyl acetate (300 mL), washed with saturated, aqueous Na₂CO₃solution (2×100 mL) and saturated, aqueous NaHCO₃ solution (2×100 mL).The organic phase was dried over Na₂SO₄ and volatiles were removed underreduced pressure to obtain crude amine intermediate (3.22 g).

The amine intermediate (3.22 g) was dissolved in DCM (5 mL). Boc₂O (2.97g, 13.69 mmol) dissolved in DCM (5 mL) and DIPEA (3.95 mL, 22.65 mmol)were added and the mixture was agitated at RT for 30 min. Boc- andMmt-protected intermediate was purified by flash chromatography.

Yield: 3.00 g (3.79 mmol).

MS: m/z 791.4=[M+H]⁺, 519.3=[M-Mmt+H]⁺ (MW calculated=791.1).

0.4 M aqueous HCl (48 mL) was added to a solution of the Boc- andMmt-protected intermediate in acetonitrile (45 mL). The mixture wasdiluted with acetonitrile (10 mL) and stirred for 1 h at RT.Subsequently, the pH value of the reaction mixture was adjusted to 5.5by addition of an aqueous 5 M NaOH solution. Acetonitrile was removedunder reduced pressure and the aqueous solution was extracted with DCM(4×100 mL). The combined organic phases were dried over Na₂SO₄ andvolatiles were removed under reduced pressure. Crude amine 14 was usedwithout further purification.

Yield: 2.52 g (3.19 mmol). A MW of 791.1 g/mol of crude amine 14 wasassumed

MS: m/z 519.3=[M+H]⁺ (MW calculated=519.8 g/mol).

Example 17 Synthesis of Linker Building Blocks 15a, 15b, and 15c

Linker building block 15a was synthesized according to the followingscheme:

Amine 14 (503 mg, 0.635 mmol, assuming a MW of 791.1 g/mol of crude 1)was dissolved in 4 mL DMF (anhydrous, mol. sieve). Fmoc-N-Me-Ala-OH (310mg, 0.953 mmol), COMU (408 mg, 0.953 mmol) and DIPEA (332 μl, 1.906mmol) were added and the reaction was allowed to stir for 3 h at RT. 150μl piperidine and 150 μl DBU were added to the mixture and stirring wascontinued for further 60 min. 400 μl acetic acid were added and productwas purified by HPLC. HPLC fractions containing product 15a wereneutralized with a saturated NaHCO₃ solution and extracted twice withDCM. Combined organic phases were dried over Na₂SO₄ and volatiles wereremoved under reduced pressure.

Yield: 203 mg (0.336 mmol).

MS: m/z 604.1=[M+H]⁺ (MW calculated=603.9 g/mol).

Linker Building Block 15b

Linker building block 15b was synthesized as described for 15a exceptthat Fmoc-Aib-OH was used instead of Fmoc-N-Me-Ala-OH.

Yield: 95 mg (0.161 mmol).

MS: m/z 604.2=[M+H]⁺ (MW calculated=603.9 g/mol).

Linker Building Block 15c

Linker building block 15c was synthesized as described for 15a exceptthat Fmoc-N-Me-Aib-OH was used instead of Fmoc-N-Me-Ala-OH.

Yield: 149 mg (0.241 mmol).

MS: m/z 619.0=[M+H]⁺ (MW calculated=617.9 g/mol).

Example 18 Synthesis of Treprostinil-Linker Thiols 16a, 16b, 16c, 16d,16e and 16f

Treprostinil-linker thiols 16a/16b were synthesized according to thefollowing scheme:

A 10 mL single use syringe reactor equipped with a PE frit was loadedwith 2-chlorotrityl chloride (TCP) resin (153 mg, loading 1.22 mmol/g,0.186 mmol). A solution of treprostinil (54 mg, 0.138 mmol) and DIPEA(60 μl, 0.346 mmol) in DCM (anhydrous, mol. sieve) was drawn into thereactor. Reactor was agitated for 2 h at RT. 200 μl methanol were addedand reactor was agitated for further 10 min. Solution was dispelled andresin was washed with DCM (5×), DMF (5×) and DCM (10×). Resin was driedunder vacuum (1 mbar). Based on weight, a treprostinil loading of 0.72mmol/g TCP resin was obtained.

900 μl THF (anhydrous, mol. sieve) and 300 μl of a 1 M LiOEt solution inTHF (300 μmol) were drawn to 30 mg treprostinil loaded TCP resin (21.6μmol) in a single use 2 mL syringe reactor equipped with a PE frit.Reactor was agitated for 40 min at RT. Solution was dispelled and resinwas washed with THF (2×). A solution of bis(pentafluorophenyl)carbonate(100 mg, 254 μmol) in 1 mL THF was drawn into the syringe which wasagitated for 90 min at RT. Solution was dispelled and resin was washedwith THF (5×) and DMF (5×). A solution of linker building block 15a (50mg, 83 μmol), DIPEA (50 287 μmol) and DMAP (1 mg, 8 μmol) in 300 μl DMF(anhydrous, mol. sieve) was drawn into the syringe. Syringe was agitatedfor 3 h at RT. Solution was dispelled and resin was washed with DMF(10×) and DCM (10×). Product was cleaved from resin by incubation with500 μl of cleavage cocktail HFIP/DCM/TES 90/10/2 v/v/v for 10 min (3×).Resin was washed with 500 μl DCM (2×). TFA (250 μL) was added to thecombined cleavage and washing solutions and the mixture was incubated atRT for 10 min. Volatiles were removed under reduced pressure. Residuewas subjected to HPLC purification which gave thiols 16a/16b as amixture of the two regioisomers. HPLC eluate was used in the next stepwithout further processing.

MS: m/z 678.1=[M+H]⁺ (MW calculated=678.0 g/mol).

Treprostinil Linker Thiols 16c/16d

Treprostinil linker thiols 16c/16d were synthesized as described for16a/16b except that linker building block 15b was used instead of 15a.Thiols 16c/16d were obtained as a mixture of isomers. HPLC eluate wasused in the next step without further processing.

MS: m/z 678.1=[M+H]⁺ (MW calculated=678.0 g/mol).

Treprostinil Linker Thiols 16e and 16f

Treprostinil linker thiols 16e and 16f were synthesized as described for16a/16b except that linker building block 15c was used instead of 15a.Two isomers assigned to structures 16e and 16f were separated by HPLC.HPLC eluates were used in the next step without further processing.

15e MS: m/z 693.0=[M+H]⁺ (MW calculated=692.0 g/mol).

15f MS: m/z 693.0=[M+H]⁺ (MW calculated=692.0 g/mol).

Example 19 Synthesis of Linker Building Blocks 17a and 17b

Linker building blocks 17a and 17b were synthesized according to thefollowing scheme:

L-Fmoc-Dpr(Boc)-OH (100 mg, 0.234 mmol) was dissolved in 0.5 mL DMF(anhydrous, mol. sieve). 6-(S-Tritylsulfanyl)-hexaneamine (71 mg, 0.189mmol), COMU (97 mg, 0.227 mmol) and DIPEA (66 μl, 0.378 mmol) were addedand mixture was stirred for 1 h at RT. Piperidine (50 μl, 0.505 mmol)and DBU (40 μl, 0.336 mmol) were added and stirring was continued for 10h. cis-Cyclohexanedicarboxylic anhydride (600 mg, 3.89 mmol) was addedand stirring was continued for 1 h. Solution was quenched withwater/acetonitrile and acidified with acetic acid Building blocks werepurified by RP-HPLC. Structures assignment of the earlier elutingdiastereomer 17a and the later eluting diastereomer 17b was donearbitrarily and could also be reverse.

Yield: 17a 30 mg (0.042 mmol), 17b 42 mg (0.059 mmol)

MS: m/z 716.2=[M+H]⁺ (MW calculated=716.0 g/mol).

Example 20 Synthesis of Treprostinil Linker Thiols 18a/18b

Linker building block 17a (11 mg, 12 μmol), EDC HCl (7.4 mg, 38.5 μmol)and DMAP (4.7 mg, 38.5 μmol) were dissolved in 300 μl DCM (anhydrous,mol. sieve). Solution was drawn to 15 mg treprostinil loaded TCP resin(10.8 μmol, 0.72 mmol/g see Example 3) in a single use 2 mL syringereactor equipped with a frit. Reactor was agitated for 15 h at RT.Solution was dispelled and resin was washed with DCM (10×). Product wascleaved by incubating resin with 500 μl HFIP/DCM 30/70 v/v for 10 min(3×). Resin was washed with 500 μl DCM (2×). To the combined cleavageand washing solutions were added 250 μl TFA and the mixture wasincubated at RT for 10 min. Volatiles were removed under reducedpressure. Residue was subjected to RP-HPLC purification which gavethiols 18a/18b as a mixture of the two regioisomers. HPLC eluate wasused in the next step without further processing.

Yield: 18a/18b 1.5 mg (2 mmol) as determined by thiol quantification byEllman Test.

MS: m/z 746.2=[M+H]⁺ (MW calculated=746.0 g/mol).

Example 21 Synthesis of PEG-Linker-Drug Conjugates 19a/b, 19c/6d, 19e,19f and 19g/19h

PEG-Linker-Drug Conjugates were Prepared According to the FollowingScheme:

To HPLC eluates of treprostinil linker thiols 16a/16b, 16c/16d, 16e, 16fand 18a/18b was given an excess of linear PEG 5 kDa maleimide. Mixtureswere neutralized by addition of pH 7.4 buffer (0.5 M phosphate) andincubated at RT. After complete consumption of thiol (approx. 1 h)mixtures were acidified with acetic acid and separated from excessPEG-maleimide by RP-HPLC. HPLC eluates were lyophilized to yieldPEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19hrespectively.

Example 22 Determination of Drug Release Half Life Time from PEGConjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h

PEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h weredissolved in pH 7.4 buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05%Tween-20, 1 mL) and incubated at 37° C. At various time points aliquotswere analyzed by UPLC to determine the amount of released treprostinilwhich was plotted against time. Drug release was found to follow firstorder kinetics. Curve fitting software was used to determine half lifetime of drug release from the respective conjugates (Table 1)

TABLE 2 entry PEG-linker-drug conjugate drug release half life time 119a/b 31 d 2 19c/19d 17 d 3 19e 24 d 4 19f 37 d 5 19g/19h 35 min

Example 23 Synthesis of Intermediate 20

The amino group of 6-(S-Tritylsulfanyl)-hexaneamine was Tmob(2,4,6-Trimethoxybenzyl) protected by dropwise addition of a solution of2,4,6-trimethoxybenzaldehyde (4.22 g, 21.51 mmol) in 88 mL methanol/DCM1/1 (v/v) to 6-(S-tritylsulfanyl)-hexaneamine (6.74 g, 17.95 mmol) andsodium cyanoborohydride (1.58 g, 25.14 mmol) in 44 mL methanol. Themixture was stirred for 1.5 h at RT and quenched with 95 mL of 0.4 Naqueous HCl solution. After further stirring at RT for 30 min mixturewas extracted with ethyl acetate (4×). Combined organic layers werewashed with sat. aqueous NaHCO₃ solution (2×) and brine. The organiclayer was dried over Na₂SO₄ and the solvent was removed under reducedpressure.

Tmob protected amine 20 was purified by flash chromatography elutingwith DCM/methanol containing 0.1% (v/v) triethylamine

Yield: 5.88 g (55%).

MS: m/z 556.3=[M+H]⁺ (MW calculated=555.79 g/mol).

Example 24 Synthesis of Intermediate 21

(1R,2S)-Cyclohexanedicarboxylic acid 1-methyl ester, CAS no. 88335-92-6(for synthesis see R. Manzano et al. J. Org. Chem. 2010, 75(15),5417-5420) (506 mg, 2.72 mmol) was dissolved in toluene (11 ml,anhydrous). Thionyl chloride (1.09 mL, 15.0 mmol) was added and mixturewas heated for 1 h at 60° C. in a pressure tube. Volatiles were removedin vacuo. A solution of Tmob protected amine 20 (1.66 g, 2.99 mmol) andDIPEA (1.12 mL, 6.43 mmol) in DCM (30 mL, anhydrous) was added andmixture was stirred for 2 h at RT. Ethyl acetate was added and theorganic layer was washed with 0.1 N aqueous HCl (2×). The organic layerwas dried over Na₂SO₄ and the solvent was removed under reducedpressure. Methyl ester 21 was purified by flash chromatography elutingwith ethyl acetate/heptane.

Yield: 1.55 g (79%).

MS: m/z 746.1=[M+Na]⁺ (MW calculated=723.98 g/mol).

Example 25 Synthesis of Intermediate 22

Methyl ester 21 (3.12 g, 4.31 mmol) was dissolved in isopropanol (10ml). 35 mL of a 1 M aqueous LiOH solution were added and the mixture wasstirred for 5 d at RT. Ethyl acetate was added and the organic layer waswashed with 0.05 N aqueous HCl (2×) and brine. The organic layer wasdried over Na₂SO₄ and the solvent was removed under reduced pressure. 22was purified by flash chromatography eluting with ethyl acetate/heptanecontaining 0.1% formic acid (v/v).

Yield: 2.41 g (79%).

MS: m/z 710.1=[M+H]⁺ (MW calculated=709.95 g/mol).

Example 26 Synthesis of Intermediate 6a

Compound 22 (1.23 g, 1.74 mmol) was dissolved in DCM (18 ml). TFA (2 mL)and TES (600 μl) were added and the mixture was stirred for 40 min atRT. Volatiles were removed in vacuo. The residue was dissolved in DCM(20 mL) and tritylchloride (728 mg, 2.61 mmol) was added. The mixturewas stirred for 2 h at RT. DCM was removed under reduced pressure.Carboxylic acid 6a was purified by flash chromatography using ethylacetate/heptane containing 0.1% formic acid (v/v) as eluent, followed byRP-HPLC purification.

Yield: 615 mg (67%).

MS: m/z 552.2=[M+Na]⁺ (MW calculated=529.75 g/mol).

Example 27 Synthesis of Treprostinil Linker Thiol 24a

Dmob protected treprostinil 8 (100 mg, 0.185 mmol), carboxylic acid 6a(195 mg, 0.368 mmol), EDC.HCl (72 mg, 0.376 mmol) and DMAP (43 mg, 0.352mmol) were dissolved in DCM (1.8 mL, anhydrous, mol. sieve). The mixturewas stirred at RT for 1 d. Ethyl acetate was added and the organic layerwas washed with 0.1 N aqueous HCl (3×) and brine. The organic layer wasdried over Na₂SO₄ and the solvent was removed under reduced pressure.

The residue was dissolved in HFIP (5 mL), TFA (250 μL) and TES (250 μL)and stirred for 30 min at RT. The precipitate was filtered off and thefiltrate was evaporated in vacuo.

UPLC analysis revealed a 4/1 ratio of regioisomers 24a and 24b (column:Kinetex 100×2.1 mm, 1.7 μm XB-C18 silica, pore size 100 Å, PhenomonexLtd, Aschaffenburg, Germany; flow rate 0.25 mL/min; solvent A:water+0.05% TFA (v/v), solvent B: acetonitrile+0.04% TFA; gradient:30-58% B (10 min), 58% B isocratic (10 min), 58-80% B (5 min), 80-99% (5min), wavelength 280 nm). 24a turned out to be identical with compound9x.

The residue was taken up in acetonitrile/water and 24a was purified byRP-HPLC (solvent A: H₂O+0.01% HCl, solvent B: MeCN+0.01% HCl, gradient:60-85% B over 16 min). Isomer 24a eluted first, followed by isomer 24b.Fractions containing pure 24a were combined and lyophilized. Mixedfractions containing 24a and 24b were subjected to repurification.

Yield 24a: 29.5 mg (24%)

MS: m/z 660.3=[M+H]⁺ (MW calculated=659.9 g/mol).

¹H-NMR (CDCl₃, δ[ppm]): 7.07 (t, 1H), 6.80 (d, 1H), 6.71 (d, 1H), 5.86(bs, 1H), 4.78-4.63 (m, 3H), 3.53 (bs, 1H), 3.14-3.03 (m, 1H), 3.03-2.83(m, 2H), 2.82-2.66 (m, 2H), 2.66-2.58 (m, 1H), 2.58-2.46 (m, 4H),2.46-2.31 (m, 1H), 2.31-2.13 (m, 1H), 2.13-1.92 (m, 2H), 1.92-1.81 (m,1H), 1.75-1.51 (m, 7H), 1.51-1.21 (m, 21H), 1.21-1.08 (m, 1H), 0.90 (t,3H).

¹³C-NMR (126 MHz, CDCl₃, δ[ppm]): 174.7, 174.2, 171.7, 155.2, 140.6,127.5, 126.3, 121.8, 109.71, 78.8, 72.4, 65.8, 48.3, 43.9, 42.5, 40.4,39.7, 37.4, 37.3, 35.4, 34.00, 33.0, 32.9, 32.1, 29.3, 28.4, 28.1, 27.0,26.4, 25.5, 24.6, 24.2, 23.2, 22.8, 14.2.

Example 28 PEGylation Reaction of Treprostinil Linker Thiol 24a with4-Arm PEG 20 kDa Maleimide

A solution of treprostinil linker thiol 24a (7.5 mg, 11.3 mmol) in 2 mLof acetonitrile/water 9/1 (v/v) was mixed with a solution of 4-arm PEG20 kDa maleimide (53.5 mg, 2.54 mmol) in 2 mL of acetonitrile/water 1/1(v/v). The pH was adjusted to 7.0 by addition of pH 7.4 buffer (50 mMphosphate, 0.8 mL). The reaction mixture was stirred at RT for 1.5 h andthen purified by RP-HPLC (solvent A: H₂O with 0.01% HCl, solvent B: MeCNwith 0.01% HCl, gradient: 45-85% B over 16 min). Product containingfractions were pooled and acetonitrile was removed under reducedpressure. The solution was neutralized by addition of pH 7.4 buffer(phosphate, 0.5 M). The solution was concentrated and the buffer wasexchanged with 10 mM pH 7.0 phosphate containing 46 g/l mannitol byultrafiltration (Vivaspin centrifugal concentrator, PES membrane with 10kDa cut off) to obtain 8.5 mL of the final solution of 25. UPLC and SECanalysis revealed a uniform material. The concentration was determinedby quantification of treprostinil content after basic hydrolysis: 30 μLaliquots were treated with 35 μL 0.5 M NaOH. After 30 min incubation atRT 35 μL acetic acid was added. The trerostinil content was determinedby UPLC by using a treprostinil calibration curve. A total treprostinilcontent of 2.0 mg was found, corresponding to 30 mg 25. Yield: 50% basedon PEG starting material.

Example 29 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compound 25

Treprostinil release kinetics from 25 was determined as described inexample 15 and compared with the results obtained from compound 13. Nodifference in half life time (5 d) was observed.

Example 30 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compound 13 in Rat Plasma

150 μl of a pH 7.5 HEPES buffer (1 M HEPES, 3 mM EDTA) were mixed with1.2 mL rat plasma (WISTAR rat Li heparin plasma, Innovative Research,Novi, Mich., USA). 150 μl of a of TransCon PEG linker treprostinil 13solution (0.15 mg 13 in 1.5 mL 10 mM phosphate 46 g/l mannitol buffer pH7.0) were added. A pH of 7.4 of the mixture was confirmed by means of apH electrode. Mixture was incubated at 37° C. At given time points 100μl aliquots were withdrawn. 100 μl aliquots were analyzed for releasedand total treprostinil content.

For analysis of released treprostinil, 100 μl aliquots were spiked with20 μl internal standard (2.8 μg/mL tolbutamide in methanol/water 1/1(v/v)) and transferred to a Ostro 96 well plate (Waters GmbH, Eschborn,Germany). Plasma proteins were precipitated by addition of three volumesof pre-cooled (0-5° C.) acetonitrile containing 1% formic acid. Positivepressure was applied (4 bar, Waters Positive Pressure-96 Processor) andeluate was lyophilized. Lyophilizate was dissolved in 40 μl of 10 mMammonium formiate pH 4.0/acetonitrile 7/3 (v/v). Solution wascentrifuged and supernatant was assayed for released treprostinil byUPLC-MS/MS.

For analysis of total treprostinil content (sum of released and carrierbound treprostinil), 100 μl aliquots were spiked with 20 μl internalstandard (2.8 μg/mL tolbutamide in methanol/water 1/1 (v/v)) and 50 μlof 0.5 M LiOH were added. Mixture was incubated in a shaker for 2 h atroom temperature. After addition 25 μL 1 M HCl the mixture wastransferred to a Ostro 96 well plate (Waters GmbH, Eschborn, Germany).Plasma proteins were precipitated by addition of three volumes ofpre-cooled (0-5° C.) acetonitrile containing 1% formic acid. Positivepressure was applied (4 bar, Waters Positive Pressure-96 Processor) andeluate was lyophilized. Lyophilizate was dissolved in 100 μl of 10 mMammonium formiate pH 4.0/acetonitrile 7/3 (v/v). Solution wascentrifuged and supernatant was assayed for total treprostinil contentby UPLC-MS/MS.

UPLC-MS/MS Method for Determination of Treprostinil Content:

The quantification of plasma treprostinil concentrations were carriedout using a Waters Acquity UPLC coupled to a Thermo LTQ OrbitrapDiscovery mass spectrometer via an ESI probe and with Waters BEH C18(50×2.1 mm I.D., 1.7 μm particle size) as analytical column (mobilephase A: 10 mM ammonium formate pH 4.6, mobile phase B: methanol, T=22°C.). The gradient system comprised a linear gradient from 0.1% B to 95%B in 4 min, an isocratic washing phase with 95% B (0.5 min), and areconditioning phase (2.4 min) with a flow rate of 0.25 mL/min.Detection of the ions was performed in the selected reaction monitoring(SRM, negative ionization) mode, monitoring the transition pairs at them/z 389.2 precursor ions to the m/z 331.2 product ions for treprostiniland m/z 269.1 precursor ions to the m/z 170.0 product ions for theinternal standard (IS) tolbutamide.

The calibration curve was acquired by plotting the extracted peak arearatio area_(treprostinil)/area_(tolbutamide) against the nominaltrepostinil concentrations of calibration standards. The results werefitted to linear regression using standard software.

The extracted peak area ratio area_(treprostinil)/area_(tolbutamide) ofthe quantification experiments at different time points were used tocalculate the treprostinil content according to the calibration curve.

Treprostinil release at time points was expressed as % treprostinilrelease compared to total treprostinil content (see FIG. 1). By using afirst order kinetics fit a half life of 4.5 d for the release kineticsof treprostinil from 25 in buffered rat plasma at 37° C. was obtained,which is in good agreement to release kinetics in pH 7.4 buffer at 37°C. (Example 15).

Example 31 PK of PEG Treprostinil Conjugate 25 in Monkeys

25 (3 mg/mL in 10 mM pH 7.0 phosphate, 46 g/L mannitol) was given at adose level of 0.5 mg/kg as a single dose by sc and iv injection in threemale cynomolgus monkeys each. Blood samples were collected at given timepoints over two weeks. The plasma was assayed for PEG content and totaltreprostinil content (sum of released and carrier bound treprostinil).Due to the fast elimination of free treprostinil compared to carrierbound treprostinil, treprostinil plasma levels reflect the presence oftreprostinil conjugate rather than free treprostinil levels.

For the analysis of total treprostinil content, 100 μL plasma samplesand treprostinil standards in cynomolgus monkey plasma were spiked with20 μL internal standard (2.8 μg/mL tolbutamide in methanol/water 1/1(v/v)) and 50 μl of 0.5 M LiOH were added. The mixture was incubated ina shaker at RT for 2.5 h. After addition of 25 μL 1 M HCl the mixturewas transferred to an Ostro 96 well plate (Waters GmbH, Eschborn,Germany). Plasma proteins were precipitated by addition of three volumesof pre-cooled (0-5° C.) acetonitrile containing 1% formic acid. Positivepressure was applied (4 bar, Waters Positive Pressure-96 Processor) andthe eluate was lyophilized. The lyophilizate was dissolved in 100 μl of10 mM ammonium formiate pH 4.0/acetonitrile 7/3 (v/v). The solution wascentrifuged and the supernatant was assayed for total treprostinilcontent by UPLC-MS/MS.

UPLC-MS/MS Method for Determination of Treprostinil Content:

The quantification of plasma treprostinil concentrations were carriedout using a Waters Acquity UPLC coupled to a Thermo LTQ OrbitrapDiscovery mass spectrometer via an ESI probe and with a Waters BEH C18(50×2.1 mm I.D., 1.7 μm particle size) as analytical column (mobilephase A: 10 mM ammonium formate pH 4.6, mobile phase B: methanol, T=22°C.). The gradient system comprised a linear gradient from 0.1% B to 95%B in 4 min, an isocratic washing phase with 95% B (0.5 min), and areconditioning phase (2.4 min) with a flow rate of 0.25 mL/min.Detection of the ions was performed in the selected reaction monitoring(SRM, negative ionization) mode, monitoring the transition pairs at them/z 389.2 precursor ions to the m/z 331.2 product ions for treprostiniland m/z 269.1 precursor ions to the m/z 170.0 product ions for theinternal standard (IS) tolbutamide.

The calibration curve was acquired by plotting the extracted peak arearatio area_(treprostinil)/area_(tolbutamide) against the nominaltrepostinil concentrations of calibration standards which were preparedin cynomolgus monkey plasma. The results were fitted to a linearregression using standard software.

The extracted peak area ratio area_(treprostinil)/area_(tolbutamide) ofthe quantification experiments at different time points were used tocalculate the treprostinil content according to the calibration curve.

For analysis of total PEG content, plasma samples underwent basicpreincubation in order to generate a uniform PEG material from 25. Thiswas based on the fact that after injection of 25 different treprostinilcarrier species are generated due to the sequencial release of 4treprostinils from carrier molecule over time.

50 μl plasma samples and 25 PEG treprostinil conjugate standards incynomolgus monkey plasma were diluted with 50 μL of 200 mM HEPESsolution (pH 7.5) and 50 μl of 0.5 M LiOH were added. Mixture wasincubated in a shaker for 2 h at room temperature. After addition of 50μL 1 M HCl the mixture was assayed using the high sensitivity PEG ELISAkit P-0003 from Life Diagnostics Inc. West Chester, Pa., USA, accordingto the manufacturer's instructions.

The calibration curve was acquired by plotting the absorption values at450 nm against the nominal PEG concentrations of calibration standards.The results were fitted to a sigmoidal curve using standard software.

The absorption values at 450 nm of the quantification experiments atdifferent time points were used to calculate the PEG content accordingto the calibration curve.

Result: Total treprostinil content analysis after a single dose scinjection of 25 reveals a prolonged duration of circulation oftreprostinil conjugate for more than two weeks in monkeys. (FIG. 2)

Single dose iv injection of 25 and plasma analysis for totaltreprostinil content revealed similar duration of circulation (FIG. 3).An apparent first order total treprostinil elimination half life time of2.9 days (rate constant k_(apparent): 0.239 d⁻¹) was obtained by usingstandard software.

In contrast plasma analysis for PEG carrier content revealed a muchslower elimination (FIG. 3). By fitting as a first order kinetics usingstandard software an PEG carrier elimination half life time of 6.6 d(rate constant k_(PEGelim): 0.105 d⁻¹) was obtained.

Equal elimination rate constant of different treprostinil carrierspecies e.g. generated by sequential linker hydrolysis/release of one tofour treprostinils from PEG carrier are assumed.

The apparent faster elimination half life time of total treprostinilcompared to PEG carrier is based on the combination of elimination ofthe PEG carrier and treprostinil release by linker hydrolysis. From thedetermined rate constant values of k_(apparent) (0.239 d⁻¹) andk_(PEGelim) (0.105 d⁻¹) a first order treprostinil release by linkercleavage rate constant k_(linker) can be calculated:

exp(−k _(apparent) t)=exp(−k _(PEGelim) t)*exp(−k _(linker) t)=exp(−t[k_(PEGelim) +k _(linker)])

After logmarithizing and rearrangement k_(linker) can be calculatedaccording to:

k _(linker) =k _(apparent) −k _(PEGelim) ; k _(linker)=0.239 d⁻¹−0.105d⁻¹=0.134 d⁻¹

By help of the equation t_(half life time)=ln(2)/k the half life time oftreprostinil release by linker hydrolysis was calculated as 5.2 d, whichis in good agreement with the 5 d linker treprostinil release half lifetime determined in vitro.

Example 32 PK of PEG Treprostinil Conjugate 25 and Free Treprostinil inRats

25 (3 mg/mL buffer (10 mM pH 7.0 phosphate, 46 g/L mannitol)) wasinjected at a dose level of 5.5 mg/kg as a single dose in male Wistarrats each. Three animals received sc injection and three animalsreceived iv injection. Blood samples were collected at given time pointsover two weeks. Blood samples (250 μL) were given directly intocollection tubes containing 50 μL acidic citrate buffer (0.5 M sodiumcitrate, pH 4.0). The plasma was assayed for free treprostinil contentand total treprostinil content (sum of free and carrier boundtreprostinil).

For the analysis of free treprostinil, 50 μL plasma were thawed on iceand mixed with 5 μl acidic citrate buffer and 10 μL internal standard(0.28 μg/mL tolbutamide in methanol/water 1/1 (v/v)). Samples weretransferred to Ostro 96 well plates (Waters GmbH, Eschborn, Germany),and plasma proteins were precipitated by rapid addition of 400 μLpre-cooled (0-5° C.) acetonitrile containing 1% formic acid. Positivepressure was applied (4 bar, Waters Positive Pressure-96 Processor) andthe eluates were collected. Subsequently, the well plates were rinsedtwo times with 100 μL ice-cold acetonitrile containing 1 vol. % formicacid. The eluates were transferred into 2 mL vials, placed into anEppendorf Thermomixer (at 10° C.) and eluates were concentrated under asoft stream of nitrogen over 45 min to a final volume of 60-80 μL. 30 μlsolvent mixture (10 mM aqueous ammonium formiate adjusted to pH 4.0 withformic acid/acetonitrile 7/3 (v/v)) were added to each vial and thesolutions were analyzed by UHPLC-MS/MS.

For preparation of calibration standards, blank plasma samples werespiked with treprostinil and treated likewise.

UHPLC-MS/MS Method for Determination of Free Treprostinil Content:

The quantification of plasma treprostinil concentrations were carriedout using an Agilent 1290 UHPLC coupled to an Agilent Triplequad 6460system (MassHunter Xcalibur software) in the ES− mode. As analyticalcolumn a Waters BEH C18 was used (50×2.1 mm I.D., 1.7 jam particle size.Mobile phase A: 10 mM ammonium formate pH 5.7, mobile phase B: methanol.The gradient system comprised a linear gradient from 35% B to 99% B in 8min, an isocratic washing phase with 99% B (0.9 min), and areconditioning phase (3 min) with a flow rate of 0.200 mL/min (T=40°C.).

Detection of the ions was performed in the SRM mode, monitoring thetransition pairs at the m/z 389.1 precursor ions to the m/z 331.1product ions for treprostinil and m/z 269.0 precursor ions to the m/z169.9 product ions for the internal standard (IS) tolbutamide.

The calibration curve was acquired by plotting the extracted peak arearatio area treprostinil/area tolbutamide against the nominal trepostinilconcentrations of calibration standards. The results were fitted to alinear regression using standard software.

The extracted peak area ratio area treprostinil/area tolbutamide of thequantification experiments at different time points were used tocalculate the treprostinil content according to the calibration curve.

Total treprostinil plasma content was determined as given in Example 31.

Result: Free and total treprostinil content analysis after a single dosesc injection of 25 reveals a prolonged circulation of treprostinilconjugate and a burstless release of free treprostinil for more thanfour days in rats after iv (FIG. 4) or sc injection (FIG. 5).

Example 33 Isolation of Intermediate 6a by Enantioseparation of RacemicMixture 6a/6b

Racemic mixture 6a/6b (107 g) was separated on a Chiralpak IA column(250×76 mm, 20 μm, flow rate 270 mL/min) using acetonitrile/acetic acid1000/1 (v/v) as eluent. Combined eluates of second eluting enantiomer(6a) were mixed with 5 vol % water and evaporated under reducedpressure. The residue was taken up in DCM (500 mL) and extracted with0.1 M HCl (500 mL, 2×) and brine (500 mL). The organic phase was driedover Na₂SO₄ and the solvent was removed under reduced pressure.

Yield: 28.9 g (27%).

MS: m/z 552.2=[M+Na]⁺ (MW calculated=529.75 g/mol).

Enantiomeric ratio of 6a/6b as determined by Chiralpak IC column(4.5×250 mm, 5 μm, eluent acetonitrile/acetic acid 1000/1 (v/v), flowrate 1 mL/min, 230 nm): 97.5/2.5

Example 34 Improved Synthesis of Treprostinil Linker Thiol 24a

Dmob protected treprostinil 8 (200 mg, 0.370 mmol), carboxylic acid 23(294 mg, 0.555 mmol), EDC.HCl (248 mg, 1.295 mmol) and DMAP (158 mg,1.295 mmol) were dissolved in DCM (2.9 mL, anhydrous, mol. sieve). Themixture was stirred at RT for 1 d. Ethyl acetate was added and theorganic layer was washed with 0.1 N aqueous HCl (3×) and brine. Theorganic layer was dried over Na₂SO₄ and the solvent was removed underreduced pressure.

The residue was dissolved in HFIP (8 mL). After addition of TFA (200 μL)and TES (200 μL) the mixture was stirred for 30 min at RT. The solutionwas extracted with heptane (16 mL, 6×) and diluted with DCM (16 mL).Solution was extracted with water (16 mL, 3×). Combined water phaseswere back extracted with DCM (8 mL). The combined DCM phases wereevaporated under reduced pressure.

UPLC analysis revealed a 9/1 ratio of regioisomers 24a and 24b (column:Kinetex 100×2.1 mm, 1.7 μm XB-C18 silica, pore size 100 Å, PhenomonexLtd, Aschaffenburg, Germany; flow rate 0.25 mL/min; solvent A:water+0.05% TFA (v/v), solvent B: acetonitrile+0.04% TFA; gradient:30-58% B (10 min), 58% β isocratic (10 min), 58-80% B (5 min), 80-99% (5min), wavelength 280 nm).

The residue was taken up in acetonitrile/water and 24a was isolated byRP-HPLC (solvent A: H₂O+0.01% HCl, solvent B: MeCN+0.01% HCl, gradient:57-62% B over 15 min). Mixed fractions were subjected to repurification.Fractions containing pure 24a were combined and lyophilized.

Yield 24a: 98 mg (39%)

MS: m/z 660.3=[M+H]⁺ (MW calculated=659.9 g/mol).

ABBREVIATIONS

-   AcOH acetic acid-   AIB 2-Aminoisobutyric acid-   BnBr benzylbromide-   Boc tert-Butoxycarbonyl--   BSA N,O-Bis-(trimethylsilyl)-acetamide-   COMU    (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium    hexafluorophosphate-   d day-   DIPEA diisopropylethylamine-   DCM dichloromethane-   DMAP 4-(Dimethylamino)pyridine-   DMF N,N-Dimethylformamide-   Dmob 2,4-dimethoxybenzyl

Abbreviations

-   DMSO dimethyl sulfoxide-   Dpr 2,3-Diaminopropionic acid-   EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide-   EDTA ethylenediamine tetraacetic acid disodium salt dihydrate-   EtOAc ethyl acetate-   eq equivalent-   h Hour-   HFIP 1,1,1,3,3,3-Hexafluoroisopropanol-   HPLC high performance liquid chromatography-   LC/MS mass spectrometry-coupled liquid chromatography-   Mal maleimido-   MeOH methanol-   MeCN acetonitrile-   min Minute-   Mmt 4-Methoxytriphenylmethyl-   mol. Molecular-   m/z Mass/charge-   NaOH Sodium hydroxide-   NHS N-hydroxysuccinimide-   PEG Polyethylene glycol-   Pfp Pentafluorophenyl-   PyBOB Benzotriazol-1-yl-oxytripyaolidinophosphonium    hexafluorophosphate-   PP polypropylene-   RT room temperature-   RP reversed phase-   sat. saturated-   soln. solution-   T temperature-   T3P propyl phosphonic anhydride-   TCP 2-Chlorotrityl chloride resin-   TES Triethylsilane-   Trt Trityl

Abbreviations

-   Tmob 2,4,6-trimethoxybenzyl-   TMS trimethylsilyl-   TransCon transiently conjugated-   THF tetrahydrofuran-   TFA trifluoroacetic acid-   UPLC ultra performance liquid chromatography-   UV ultra violet

While this invention has been described in conjunction with the specificembodiments outlined above, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart. Accordingly, the preferred embodiments of the invention as setforth above are intended to be illustrative, not limiting. Variouschanges may be made without departing from the spirit and scope of theinventions as defined in the following claims.

1. A pharmaceutical composition comprising a prostacyclin compound, andoptionally one or more pharmaceutically acceptable excipients, whereinthe concentration of the prostacyclin compound is sufficient to maintaina therapeutically effective level of prostacyclin in blood plasma for atleast 12 hours after a single subcutaneous or intramuscular injection.2. The pharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises the prostacyclin compound in a concentration of atleast 0.05 mg/ml.
 3. The pharmaceutical composition comprising aprostacyclin compound of claim 1 in a concentration of at least 0.05mg/ml, and wherein a single dose of the pharmaceutical compositioncomprises at least 0.05 mg of the prostacyclin compound.
 4. Thepharmaceutical composition of claim 1, wherein the time period betweenadministration of a pharmaceutical composition is at least about 12hours.
 5. A pharmaceutical composition comprising a prostacyclincompound, and optionally one or more pharmaceutically acceptableexcipients, wherein the pharmaceutical composition is characterized byhaving a pharmacokinetic profile in vivo in a mammal, in particular in ahuman, with substantially no burst of the prostacyclin compound.
 6. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is characterized by having a pharmacokinetic profile in vivoin a mammal, in particular in a human, with substantially no burst ofthe prostacyclin compound.
 7. A pharmaceutical composition comprising aprostacyclin compound, and optionally one or more pharmaceuticallyacceptable excipients, wherein the pharmaceutical composition ischaracterized by exhibiting a peak to trough ratio of the prostacyclincompound in a mammal, in particular in a human, of less than
 5. 8. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is characterized by exhibiting a peak to trough ratio of theprostacyclin compound in a mammal, in particular in a human, of lessthan
 5. 9. A pharmaceutical composition comprising a prostacyclincompound, and optionally one or more pharmaceutically acceptableexcipients, wherein the prostacyclin compound has an activity of <20% ofthe activity of free prostacyclin.
 10. The pharmaceutical composition ofclaim 1, wherein the prostacyclin compound has an activity of <20% ofthe activity of free prostacyclin.
 11. A pharmaceutical compositioncomprising a prostacyclin prodrug, and optionally one or morepharmaceutically acceptable excipients, wherein the pharmaceuticalcomposition is characterized in that after subcutaneous or intramuscularadministration of said prostacyclin prodrug more than 50% of theadministered prostacyclin dose is releasable within the bloodcompartment.
 12. The pharmaceutical composition of claim 1, wherein theprostacyclin compound is a prostacyclin prodrug characterized in thatafter subcutaneous or intramuscular administration of said prostacyclinprodrug more than 50% of the administered prostacyclin dose isreleasable within the blood compartment.
 13. The pharmaceuticalcomposition of claim 1, wherein the prostacyclin compound is in a depot.14. The pharmaceutical composition of claim 13, wherein the prostacyclincompound is covalently linked to a compound in the depot.
 15. Thepharmaceutical composition of claim 1, wherein the prostacyclin compoundis a prodrug of prostacyclin.
 16. The pharmaceutical composition ofclaim 15, wherein the promoiety of the prodrug comprises a linear orbranched PEG moiety.
 17. A pharmaceutical composition comprising aprostacyclin prodrug, wherein said pharmaceutical composition ischaracterized in that the prostacyclin compound releases prostacyclin ina plasma-independent manner.
 18. The pharmaceutical composition of claim5, characterized by being administered by injection.
 19. A method forthe treatment or prevention of pulmonary hypertension, ischemicdiseases, preferably peripheral vascular disease including peripheralarterial disease, Raynaud's phenomenon including Raynaud's disease andRaynaud's syndrome, scleroderma including systemic sclerosis, myocardialischemia, ischemic stroke, renal insufficiency, ischemic ulcersincluding digital ulcers, heart failure, in particular congestive heartfailure, portopulmonary hypertension, interstitial lung disease,idiopathic pulmonary fibrosis, conditions requiring anticoagulation, inparticular post MI, post cardiac surgery, thrombotic microangiopathy,extracorporeal circulation, central retinal vein occlusion,atherosclerosis, inflammatory diseases, in particular COPD, psoriasis,hypertension, in particular preeclampsia, reproduction and parturition,cancer or other conditions of unregulated cell growth, cell/tissuepreservation and other emerging therapeutic areas where prostacyclintreatment appears to have a beneficial role comprising the step ofadministering a pharmaceutical composition of claim
 1. 20. A method fortreating pulmonary hypertension, wherein the method comprises the stepof subcutaneous or intramuscular administration, to a patient withpulmonary hypertension, of a pharmaceutical composition comprising (a) apolymer carrier-linked prostacyclin prodrug, and optionally one or morepharmaceutically acceptable excipients, or (b) a prostacyclin compoundand at least one polymer, and optionally one or more pharmaceuticallyacceptable excipients, wherein the pharmaceutical composition releasestherapeutically effective amounts of free prostacyclin compound for aperiod of time of at least 12 hours.
 21. The method of claim 20, whereinthe pharmaceutical composition of (b) comprises from about 0.05 to about10 weight percent the prostacyclin compound and from about 0.5 to about20 weight percent total polymer content.
 22. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition ischaracterized by exhibiting a peak to trough ratio of the prostacyclincompound in a mammal, in particular in a human, of less than
 2. 23. Apharmaceutical composition comprising a prostacyclin compound, andoptionally one or more pharmaceutically acceptable excipients, whereinthe prostacyclin compound has an activity of <5% of the activity of freeprostacyclin.
 24. The pharmaceutical composition of claim 1, wherein theprostacyclin compound has an activity of <5% of the activity of freeprostacyclin.
 25. The pharmaceutical composition of claim 1, wherein theprostacyclin compound is in a hydrated polymer matrix.
 26. Thepharmaceutical composition of claim 13, wherein the prostacyclincompound is covalently linked to a well hydrated polymer matrix.
 27. Thepharmaceutical composition of claim 5, characterized by beingadministered by subcutaneous or intramuscular injection.